the synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9- dien-28

The Synthetic Triterpenoid 2-Cyano-3,12-dioxooleana-1,9-dien-28-oic Acid-Imidazolide Alters Transforming GrowthFactor �-dependent Signaling and Cell Migration byAffecting the Cytoskeleton and the Polarity Complex*□S

Received for publication, May 16, 2007, and in revised form, February 8, 2008 Published, JBC Papers in Press, February 18, 2008, DOI 10.1074/jbc.M704064200

Ciric To‡, Sarang Kulkarni§, Tony Pawson§, Tadashi Honda¶, Gordon W. Gribble¶, Michael B. Sporn�1,Jeffrey L. Wrana§2, and Gianni M. Di Guglielmo‡3

From the ‡Department of Physiology and Pharmacology, University of Western Ontario, London, Ontario N6A 5C1, Canada,the §Programme in Molecular Biology and Cancer, Samuel Lunenfeld Research Institute, Mount Sinai Hospital,Toronto, Ontario M5G 1X5, Canada, the ¶Department of Chemistry, Dartmouth College, Hanover, New Hampshire 03755,and the �Department of Pharmacology, Dartmouth Medical School, Hanover, New Hampshire 03755

The anti-tumor synthetic triterpenoid 2-cyano-3,12-di-oxooleana-1,9-dien-28-oic acid (CDDO)-imidazolide (CDDO-Im) ectopically activates the transforming growth factor �(TGF�)-Smad pathway and extends the duration of signaling byan undefined mechanism. Here we show that CDDO-Im-dependent persistence of Smad2 phosphorylation is independ-ent of Smad2 phosphatase activity and correlates with delayedTGF� receptor degradation and trafficking. Altered TGF� traf-ficking parallels the dispersal of EEA1-positive endosomes fromthe perinuclear region of CDDO-Im-treated cells. The effect ofCDDO-Im on the EEA1 compartment led to an analysis of thecytoskeleton, and we observed that CDDO-Im alters microtu-bule dynamics by disrupting the microtubule-capping protein,Clip-170. Interestingly, biotinylated triterpenoid was found tolocalize to the polarity complex at the leading edge of migratingcells. Furthermore, CDDO-Im disrupted the localization ofIQGAP1, PKC�, Par6, and TGF� receptors from the leadingedge of migrating cells and inhibited TGF�-dependent cellmigration. Thus, the synthetic triterpenoid CDDO-Im inter-feres with TGF� receptor trafficking and turnover and disruptscell migration by severing the link between members of thepolarity complex and the microtubule network.

Transforming growth factor� (TGF�)4 familymembers reg-ulate many cellular functions, including proliferation and dif-

ferentiation, and TGF� is a potent apoptotic agent in manycells, including early stage epithelial tumors (1). However, inlate stage epithelial tumors, TGF� becomes a metastatic agentand stimulates epithelial tomesenchymal transition (EMT) andcell migration (2–5). Signaling by TGF� growth factor is initi-ated via ligand-induced heteromeric complex formation of theSer/Thr kinase type I (T�RI) and type II (T�RII) transmem-brane receptors (6). The phosphorylation of receptor-regulatedSmad proteins R-Smad2 and R-Smad3 is facilitated by Smadanchor for receptor activation protein, which binds the recep-tors and recruits R-Smad to the membrane of EEA1-positiveearly endosomes (7). Early endosomes also contain other mod-ulators of TGF� receptor signaling, such as hepatocyte growthfactor-regulated tyrosine kinase substrate (8) and cytoplasmicpromyelocytic leukemia protein (9).Inactivation of the TGF� signaling pathway is carried out by

several mechanisms. Phosphorylated Smad2 is targeted by thenuclear phosphatase PPM1A (10), and the receptors are thetarget of inhibitory Smad7, which interacts with T�RI in lipidrafts/caveolae and recruits the E3 ligases, Smurf1 and Smurf2,which direct ubiquitin-dependent degradation of the TGF�receptors (11–13). Perturbation of lipid rafts increases signal-ing and reduces the rate of receptor degradation (14–18). Thus,the signal transduction pathway initiated by cell surface TGF�receptor complex is dependent on receptor internalization andtrafficking via distinct endocytic pathways (19).Endocytosis of cell surface proteins is dependent on the

microtubule cytoskeleton (20–22). Microtubules are dynamicprotein filaments that span the cell interior and provide amechanical framework for chromosome sorting, cell polarity,and organelle localization, among other functions (23–28).Microtubules grow and shrink from their plus-ends, and theirminus-ends are usually located at the microtubule-organizingcenter but are also found at the apical domain in epithelial cells(29).

* This work was supported by National Cancer Institute of Canada (NCIC)Terry Fox Foundation Grant 017189 (to G. M. D. G.). The costs of publica-tion of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Figs. 1–3 and Movies 1– 4.

1 Supported by National Institutes of Health Grant R01 CA78814, the NationalFoundation for Cancer Research, and Reata Pharmaceuticals, Inc.

2 Work in the laboratory of this author was supported by grants from the NCICand Canadian Institutes of Health Research Grant MOP-74692.

3 To whom correspondence should be addressed: Dept. of Physiology andPharmacology, Medical Sciences Bldg., University of Western Ontario, Lon-don, Ontario N6A 5C1, Canada. Tel.: 519-661-2111 (ext. 80042); E-mail:[email protected].

4 The abbreviations used are: TGF�, transforming growth factor �; CDDO,2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid; CDDO-Im, CDDO-imida-

zolide; CDDO-biotin, biotinylated CDDO; EMT, epithelial to mesenchymaltransition; T�RI and T�RII, TGF� Ser/Thr kinase type I and II membranereceptor, respectively; E3, ubiquitin-protein isopeptide ligase; HA, hemag-glutinin; GFP, green fluorescent protein; Mes, 4-morpholineethanesulfonicacid; DAPI, 4�,6-diamidino-2-phenylindole; DIC, differential interferencecontrast.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 17, pp. 11700 –11713, April 25, 2008© 2008 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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Polarized migration of cells as well as the movement of vesi-cles along the microtubules is dependent on molecular motorsandmicrotubule-binding proteins, such as the capping protein,Clip-170 (30, 31). Furthermore, the association ofmicrotubule-bound Clip-170 at the cell membrane with the Rac1/Cdc42-binding protein, IQGAP1, is an essential mediator betweenmicrotubules and the leading edge of migrating cells (32, 33).Cdc42 also binds to the Par6-PKC� polarity complex, which inturn links Cdc42 to APC and the microtubule network to forma focal point for directional cellular movement (26). Thesemolecular links, in combination with the observation that Par6associates with TGF� receptors (34), have introduced a newarea of study to the field of TGF�-dependent signaling in cellmigration and metastasis (2).Chemotherapeutic agents that block TGF�-dependent sig-

naling and TGF�-dependent metastasis have been a majorfocus in cancer chemotherapy (35). Recently, 2-cyano-3,12-di-oxooleana-1,9-dien-28-oic acid (CDDO), has been shown to bea promising cancer therapeutic agent that is currently in PhaseI clinical trials (36). Interestingly, both CDDO and its imidazol-ide derivative, CDDO-Im, have been shown to synergisticallyincrease cellular responses to factors such as TGF� in cell cul-ture studies (37–39). Themechanismwhereby CDDO-Im doesthis has yet to be elucidated. In this study, we demonstrate thatCDDO-Im alters TGF� cell signaling and receptor traffickingand inhibits cell migration by disrupting cytoskeletal attach-ments to the polarity complex.

EXPERIMENTAL PROCEDURES

Cell Lines and Antibodies

Mv1Lu cells were cultured inminimal essentialmedium sup-plemented with 1% nonessential amino acids. Mv1Lu cells sta-bly transfectedwith theHAepitope-taggedTGF� type II recep-tor (HATcells)were cultured inminimal essentialmediumplus1% nonessential amino acids and 0.3 mg/ml hygromycin.COS-7, C2C12, HEK293, and Rat2 cells were cultured in Dul-becco’s modified Eagle’s medium. All media were supple-mented with 10% fetal bovine serum unless otherwise stated.Texas Red-conjugated phalloidin, anti-caveolin-1 (610059),anti-EEA1 (610456), anti-Rac1 (610650), anti-GM130(610822), anti-Smad2 (610842), and anti-LAMP1 (555798)antibodies were purchased from BD Transduction Laborato-ries (Mississauga, Canada). Monoclonal anti-tubulin (Tub2.1),anti-FLAG (M2), and polyclonal anti-actin (A2668) antibodieswere purchased fromSigma. Polyclonal anti-PKC� (C-20), anti-TGF� type II receptor (C-16), anti-Par6 (H-90), anti-IQGAP1(H-109), anti-HA (Y-11), and goat anti-lamin A/C antibodies(sc-6215) were purchased from Santa Cruz Biotechnology, Inc.(Santa Cruz, CA). Anti-phospho-Smad2 (AB3849) antibodieswere purchased from Chemicon (Temecula, CA). The poly-clonal phospho-Par6 (Ser-345) antibody was used as previouslydescribed (34). The polyclonal anti-Clip-170 (N-terminal) anti-body was a generous gift fromDr. K. Kaibuchi (Nagoya Univer-sity, Nagoya, Japan), and the polyclonal anti-calnexin waskindly provided by Dr. J. J. M. Bergeron and P. H. Cameron(McGill University, Montreal, Canada). The GFP-tagged Clip-

170 construct was a generous gift from Dr. F. Perez (CNRS,Paris, France).

Affinity Labeling

Cells were preincubated in control media or media contain-ing 1 �M CDDO-Im for 1 h at 37 °C, placed on ice, and incu-bated with 250 pM 125I-TGF� in KRH plus 0.5% bovine serumalbumin at 4 °C for 2 h. Following cross-linking with disuccin-imidyl suberate, cells were lysed (time 0) or incubated at 37 °Cfor 2, 4, or 8 h prior to lysis. Receptors were visualized by SDS-PAGE and quantified using PhosphorImager analysis (Amer-sham Biosciences).

Subcellular Fractionation

Cytoskeleton—Fractions containing the cytoskeleton wereseparated from those containing detergent-solubilized mem-branes and cytosol by the method described by Contin et al.(40). Briefly, COS-7 cells were incubated for 3 h in controlmedium or media containing 10 �M nocodazole or 1 �MCDDO-Im and then rinsed with microtubule stabilizationbuffer (90 mM Mes (pH 6.7), 1 mM EGTA, 1 mM MgCl2, 10%(v/v) glycerol) that had been preheated to 37 °C. Cells werethen lysed with microtubule stabilization buffer containing10 �M placitaxel (Sigma), 0.5% Triton X-100, and proteaseinhibitors for 4 min at 37 °C. The solubilized fractions werethen collected. To collect the remaining cellular structurescontaining the cytoskeleton, SDS-PAGE sample buffer wasadded to the culture dishes. Following scraping and passag-ing through a syringe, the fractions containing the cytoskel-eton were collected.To analyze the partitioning of cytoskeletal proteins, frac-

tions containing the soluble proteins or the cytoskeletonwere subjected to SDS-PAGE followed by immunoblottingwith anti-Rac1, anti-tubulin, anti-actin, anti-IQGAP1, anti-EEA1, anti-calnexin, anti-LAMP1, anti-GM130, or anti-Clip-170 antibodies.Nuclear Fractionation—To quantify the amount of Smad2

present in the nucleus in the presence of CDDO-Im, subcellularfractionationwas carried out as described by Cong andVarmus(41). Briefly, cells were incubated with 0.5mMTGF� for 30minin the absence or presence of 1 �M CDDO-Im, washed, andincubated in media containing Me2SO (control), 1 �M CDDO-Im, 10 �M SB431542, or both compounds (CDDO-Im �SB431542) at 37 °C for an additional 1 or 4 h. The cells werethen scraped into ice-cold PBS and collected by centrifugationat 1000 � gav for 5 min. The cells were then washed once againwith phosphate-buffered saline and resuspended in hypotonicbuffer (10 mMHepes-KOH, 10 mMNaCl, 1 mM KH2PO4, 5 mMNaHCO3, 1 mM CaCl2, 0.5 mM MgCl2, pH 7.4). After a 5-minincubation on ice, cells were homogenized with 50 strokesusing a Dounce homogenizer. Cells were then centrifuged at1000� gav for 5min. The pellets were washed twice with hypo-tonic buffer and resuspended in nuclear isolation buffer (10mMTris (pH 7.5), 300 mM sucrose, 0.1% Nonidet P-40). The pelletsin the nuclear isolation buffer were then further homogenized50 times using a Dounce homogenizer and centrifuged at1000 � gav for 5 min. The nuclear pellet was resuspended withnuclear isolation buffer with 1% Triton X-100 to generate the

CDDO-Im Alters TGF�-dependent Signaling and Cell Migration

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nuclear fraction. The nuclear fractions were subjected to SDS-PAGE and immunoblotted with mouse anti-Smad2 or goatanti-lamin A/C antibodies. The levels of Smad2 were quanti-tated usingQuantityOne software (Bio-Rad) and normalized tothe amount of lamin A/C in each fraction.Phospho-Smad2TimeCourse—Cellswere incubatedwith 0.5

mM TGF� for 30 min, in the absence or presence of 1 �M

CDDO-Im, washed, and incubated inmedia containingMe2SO(control), 1 �M CDDO-Im, 10 �M SB431542 (Sigma), or bothcompounds (CDDO-Im� SB431542) at 37 °C for an additional1 or 4 h prior to lysis. Cell lysates were then analyzed by SDS-PAGE and immunoblotted with mouse anti-Smad2 or rabbitanti-phospho-Smad2 antibodies. Quantitation of the amountof phosphorylated Smad2 was carried out using QuantityOnesoftware and normalized to Smad2 levels.

Immunofluorescence Microscopy

Smad2 Nuclear Accumulation—Cells were incubated with0.5 mM TGF� for 30 min in the absence or presence of 1 �M

CDDO-Im, washed, and incubated inmedia containingMe2SO(control), 1 �M CDDO-Im, 10 �M SB431542, or both com-pounds (CDDO-Im� SB431542) at 37 °C for an additional 1, 4,or 8 h prior to fixation and permeabilization. Cells were thenimmunostained with anti-Smad2/3 antibody followed by Cy2-conjugated secondary antibody. DAPI staining was used tovisualize cell nuclei. To quantify the amount of Smad2 nuclearlocalization, nuclear versus cytoplasmic intensity profiles weregenerated from individual cells using ImagePro software. Thequantitation of �100 cells/condition from three experimentswas graphed as the nuclear signal minus the cytoplasmic signalversus time � S.D.Receptor Traffic—Receptor internalization studies were car-

ried out as previously described (15). Briefly,HATcells express-ing extracellularly HA-tagged T�RII receptors were incubatedwith biotinylated-TGF� for 2 h at 4 °C, washed, and incubatedwith Cy3-streptavidin (Jackson Immunoresearch, West Grove,PA). Cells were then washed and incubated at 37 °C for 1 h withor without CDDO-Im. After the 37 °C incubation, cells werefixed and incubated with mouse monoclonal anti-EEA1 anti-bodies and rabbit polyclonal anti-caveolin-1 antibodies. Anti-EEA1 and anti-caveolin-1were detected using anti-mouseCy5-conjugated antibodies and anti-rabbit Cy2-conjugatedantibodies (Jackson Laboratories Inc.), respectively. Acid wash-ing was carried out as previously described (15). Images werecaptured using an Olympus 1X81 inverted microscopeequipped with fluorescence optics and deconvolved usingImagePro software.EEA1 Distribution—To visualize the effect of CDDO-Im on

EEA1-positive endosomes, we assessed the staining pattern ofthe EEA1 compartment via immunofluorescence microscopyas described abovewith the exception that Cy2-labeled second-ary antibodies were used. DAPI staining was used to visualizecell nuclei.Staining observed to be localized to an area no greater than

one-half of the cell area and located more intensely on one sideof the nucleuswas scored as “perinuclear-positive.” If the inten-sity and distribution of the stain was equal throughout the cell

body, it was scored as “dispersed.” The quantitation of �100cells/condition from three experiments was graphed � S.D.Cytoskeleton Studies—To visualize the effect of CDDO-Im

on the microtubule cytoskeleton, cells were incubated for 1 hwith or without 1 �M CDDO-Im or 10 �M nocodazole (Sigma)at 37 °C. Following fixation and permeabilization, cells wereincubated with monoclonal anti-tubulin antibody followed byfluorescein isothiocyanate-conjugated secondary antibodies.To assess the effect of CDDO-Im on the actin cytoskeleton,cells were incubated with 1 �M CDDO-Im or 10 �M cytochala-sin B for 1 h prior to fixation and permeabilization. The cellswere then incubated for 30 min with Texas Red-conjugatedphalloidin. Images were collected from a LeicaDMIRE invertedmicroscope.Scratch Assay—Rat2 fibroblasts were grown to confluence,

and the cellmonolayerwas scratchedwith a sterile pipette tip tocreate a “wound.” For bright field or DIC microscopy, imageswere collected using an Olympus IX81 inverted microscope.For immunofluorescence studies, cells were fixed, permeabi-lized, and incubated with anti-Clip-170, anti-tubulin, anti-Rac1, anti-T�RII, anti-IQGAP1, anti-Par6, or anti-PKC� anti-bodies. Following fluorescently tagged secondary antibodyincubation, the nuclei of cells were stained with DAPI, and cellswere visualized using an Olympus IX81 microscope controlledby QED in vivo software (Olympus).The quantitation of the number of cells containing proteins

at the leading edge of the cell was carried out using �100 cells/condition from three experiments� S.D. Briefly, a cell contain-ing a positive immunofluorescence signal only along the edge ofthe plasma membrane that was directly adjacent to the scratchwas scored as positive for leading edge localization. If a cellcontained no signal along the edge adjacent to the scratch orcontained dispersed signal along the complete cell periphery, itwas scored as negative for leading edge staining.Clip-170 Movies—Rat2 fibroblasts were microinjected with

0.25 nM cDNA encoding GFP-tagged Clip-170 protein andincubated at 37 °C for 4 h. The cells were then incubated incontrol medium or media containing 0.1 or 1 �M CDDO-Im.Expressed GFP-tagged Clip-170 was visualized by immunoflu-orescence, and time lapse images were collected using a LeicaDMIRE inverted microscope utilizing Openlab 3.0 software.

Cell Transfection

Cells were transfected with the constructs described in thefigure legends using the calcium phosphate method as previ-ously described (15).

Immunoprecipitation

Cells were lysed (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1mM EDTA, 0.5% Triton X-100, 1mM phenylmethylsulfonyl flu-oride, and mixture of protease inhibitors) and centrifuged at15,000 � gav at 4 °C for 5 min. Aliquots of supernatants werecollected for analysis of total protein concentration. Theremaining cell lysates were incubated with primary antibody,followed by incubation with protein G-Sepharose. The precip-itates were washed three times with lysis buffer, eluted withLaemmli sample buffer, and subjected to SDS-PAGE andimmunoblot analysis.




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CDDO-biotin Subcellular Localization

Rat2 fibroblasts were grown to 100% confluence andscratched to create a wound. The cells were then incubated at37 °C for 6 h to allow for the establishment of polarity. The cellswere then fixed, permeabilized, and incubated with Rac1 anti-bodies followed by Cy2-labeled secondary antibodies. The cellswere then incubated with 10 �M biotin, CDDO, or CDDO-biotin for 2 h, followed by incubation with Cy3-labeled strepta-vidin and DAPI. Cells were visualized using an Olympus IX81inverted microscope.

Protein Concentration

Protein concentrations of lysates were measured using theLowry method (Fisher).

RESULTS

The triterpenoid, CDDO-imidazolide (CDDO-Im) extendsTGF� signal transduction in several cell lines (37, 39); however,the mechanism(s) and functional consequences have yet to befully elucidated.In order to study the mechanism of how CDDO-Im extends

TGF�-dependent signaling, we first attempted to identify celllines in which TGF�-dependent Smad2 phosphorylation istransient. We treated various cell lines with a 30-min pulse ofTGF� and investigated the state of Smad2 phosphorylation byimmunoblot analysis using a phospho-Smad2-specific anti-body. We observed that the phosphorylation of Smad2 wastransient in Rat2, COS-7, and C2C12 cells (�4.5 h) whereas it

was prolonged (�8 h) inHepG2 andHeLa cells (Fig. 1) (data not shown).In Rat2 and COS-7 cells, Smad2phosphorylation was observed 30min after TGF� stimulation andattenuated over the remainder ofthe time course (Fig. 1, A and B,lanes 1–4). In C2C12 cells, Smad2phosphorylation returned to back-ground levels within 1 h of ligandremoval from the culture medium(Fig. 1C, lanes 1–4). Having ob-served transient Smad2 phospho-rylation in Rat2, C2C12, andCOS-7 cells, we next assessed theeffect of co-incubating cells withTGF� and CDDO-Im. We ob-served that CDDO-Im increasedboth the extent and duration ofTGF�-dependent Smad2 phos-phorylation in all of the three celltypes tested (Fig. 1, lanes 5–8).We next investigated if the effect

of CDDO-Im on the duration ofSmad2 phosphorylation could bedue to an inhibition of Smad2 phos-phatase activity. In order to test this,we employed one of the strategiesused by Lin et al. (10) to identifyPPM1A as the nuclear Smad2 phos-

FIGURE 1. CDDO-Im extends TGF�-dependent Smad2 phosphorylationindependently of Smad2 phosphatase activity. Rat2 (A), COS-7 (B), orC2C12 (C) cells were incubated with 0.5 mM TGF� for 30 min in the absence orpresence of 1 �M CDDO-Im, washed, and incubated in media containingMe2SO (DMSO) (Control), 1 �M CDDO-Im, 10 �M SB431542, or both com-pounds (CDDO-Im � SB431542) at 37 °C for an additional 1 or 4 h prior to lysis.One hundred micrograms of cell lysates were then processed for SDS-PAGEand immunoblotted with anti-phosphospecific Smad2 (�-P-S2) or Smad2/3(�-S2/3) antibodies. The bands corresponding to phosphoserine-modifiedSmad2 (P-S2) and Smad2 (S2) are indicated. Representative blots from fourexperiments are shown.

FIGURE 2. CDDO-Im extends TGF�-dependent Smad2 nuclear accumulation independently of Smad2phosphatase activity. Unstimulated C2C12 cells (A) or C2C12 cells pulsed with 0.5 mM TGF� for 30 min in theabsence or presence of 1 �M CDDO-Im were incubated in media containing Me2SO (DMSO) (B), 1 �M CDDO-Im(C), 10 �M SB431542 (D), or both compounds (E) at 37 °C for 1 or 8 h. The cells were then processed forimmunofluorescence microscopy and probed with DAPI (blue) to indicate nuclei and anti-Smad2/3 (Smad2)antibodies (green). Nuclear and cytoplasmic Smad2 from three experiments � S.D. were quantitated usingImagePro software and graphed as nuclear-cytoplasmic Smad2 versus time (F). Bar, 10 �m.




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phatase. Briefly, the TGF� type I receptor inhibitor, SB431542,was added to the cell culture media after a preincubation of thecells with TGF�. The addition of the inhibitor would allowassessment of whether TGF� receptor activity would influencethe effect of CDDO-Im on TGF�-dependent Smad2 phospho-rylation. In Rat2, COS-7, and C2C12 cells, the SB431542 inhib-itor greatly reduced the phosphorylation of Smad2within 1 h ofincubation, indicating that these cell types have functionalphosphatase activity (Fig. 1, lanes 9–12). We next assessed ifCDDO-Imwould influence Smad2 phosphatase by co-incubat-ing cells with SB431542 and CDDO-Im. We observed thatCDDO-Im did not extend TGF�-dependent Smad2 phospho-rylation in Rat2, COS-7, or C2C12 cells when SB431542 waspresent in the culture media (Fig. 1, lanes 13–16). These results

suggest that the Smad2 phosphatase was not a CDDO-Im tar-get, since CDDO-Im was unable to extend the duration ofSmad2 phosphorylation after TGF� receptor inactivation.

As a second line of investigation, we assessed TGF�-depend-ent Smad2 nuclear translocation using immunofluorescencemicroscopy (Fig. 2). We found that in the absence of TGF�,Smad2 staining was observed throughout the cytoplasm ofC2C12 cells (Fig. 2A). After 1 h of TGF� stimulation, Smad2staining was only observed in the nuclei of cells regardless oftreatment (Fig. 2, B–E, top panels). Eight hours after TGF� wasremoved from the cell culture medium, Smad2 cytoplasmicstaining reappeared in both control and CDDO-Im-treatedcells (Fig. 2, B and C, bottom panels).

Consistent with the phospho-Smad2 time course studies, theTGF� type I receptor inhibitor, SB431542, increased cytoplas-mic Smad2 staining after 8 h of incubation (Fig. 2D), and thiswas also observed if the cells were co-incubated with SB431542and CDDO-Im (Fig. 2E). These results were supported by thequantitation of three experiments (Fig. 2F), and similar resultswere obtained with the use of Rat2 or COS-7 cells (data notshown).Finally, to further evaluate TGF�-dependent nuclear accu-

mulation of Smad2 in the presence or absence of CDDO-Imand/or SB431542, we carried out subcellular fractionationstudies. We assessed TGF�-dependent Smad2 nuclear accu-mulation by immunoblotting isolated nuclear fractions withanti-Smad2 antibodies (supplemental Fig. 1). In control cells,we observed Smad2 in the nuclear fractions after 0.5 h of TGF�stimulation and the signal attenuated after 4.5 h of stimulation(supplemental Fig. 1, lanes 1–3). This was accentuated if thecellswere incubatedwithCDDO-Im (supplemental Fig. 1, lanes

FIGURE 3. CDDO-Im delays TGF� receptor degradation and trafficking.A, C2C12, COS-7, or Mv1Lu cells were affinity-labeled with 125I-TGF� at 4 °C,cross-linked, and lysed (zero time control) or incubated at 37 °C for 2, 4, or 8 hprior to lysis. One hundred micrograms of cell lysates were then subjected toSDS-PAGE followed by autoradiography and PhosphorImager analysis. Thebands representing T�RI or T�RII are indicated on the left side of each panel.Total receptor levels were quantified and graphed (right panel) as receptorlevels (percentage of control) versus time (n � 3 � S.D.). B, Mv1Lu cells stablyexpressing T�RII were incubated at 4 °C with biotinylated TGF� (bTGF�), fol-lowed by streptavidin Cy3 (red). The cells were then incubated at 37 °C in theabsence (left; Control) or presence (right) of 1 �M CDDO-Im for 1 h. Cells werethen fixed, permeabilized, and immunostained with anti-caveolin-1 (Cav-1;green) and anti-EEA1 (EEA1; blue) antibodies. Areas of interest (white boxes)are magnified for both the control and CDDO-Im-treated cells and are shownbelow each panel (insets). Representative cells shown indicate the co-local-ization of biotinylated TGF� with either caveolin-1 or EEA1 indicated byyellow or magenta arrowheads, respectively. The contour (dotted line) ofeach cell was established using bright field images and overlaid on theDAPI nuclear stain. Representative micrographs from three experimentsare shown. Bar, 10 �m.

FIGURE 4. CDDO-Im disperses the EEA1 early endosomal compartment.A, vehicle-treated Mv1Lu cells (Control) or Mv1lu cells treated with 1 �M

CDDO-Im for 1 h at 37 °C were fixed, permeabilized, and immunostained withanti-EEA1 antibodies (EEA1; green). The nuclei were visualized using DAPIstaining (blue). A representative cell (inset) was magnified and is shown at thebottom. Bar, 10 �m. B, three experiments were carried out as described in A.One hundred cells from each experimental condition were analyzed, scoredon the basis of perinuclear or dispersed staining of EEA1 protein, and graphed(n � 3 � S.D.).




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4–6). We observed that the TGF� type I receptor inhibitor,SB431542, decreased the amount of Smad2 in the nuclear frac-tions after 4 h of incubation (supplemental Fig. 1, lanes 7–9),and this was also observed if the cells were co-incubated withSB431542 and CDDO-Im (supplemental Fig. 1, lanes 10–12).Taken together, these results support the conclusion thatCDDO-Im increases TGF�-dependent Smad2 phosphoryla-tion and nuclear accumulation in a process that is independentof Smad2 phosphatase activity.CDDO-Im Interferes with TGF� Receptor Degradation and

Trafficking—Efficient phosphorylation and nuclear transloca-tion of Smad2 is dependent on the proper targeting of TGF�receptors to the early endosomal compartment (8, 9, 15,42–45). Inhibition of the signal transduction pathway isdependent on Smad2 phosphatase activity (10) and on the asso-ciation of TGF� receptors with the Smad7-Smurf2 complex,

which inhibits TGF� receptorkinase activity and promotes recep-tor complex degradation (11, 12).Since we concluded that Smad2phosphatase activity was not tar-geted by CDDO-Im, we attemptedto determine if CDDO-Im regulatesthe kinetics of Smad activation byaltering TGF� receptor degrada-tion. We therefore assessed TGF�receptor turnover in C2C12 andCOS-7 cells by affinity-labelingreceptors with 125I-TGF� at 4 °Cfollowed by incubation at 37 °C,SDS-PAGE, and PhosphorImageranalysis. We observed that bothC2C12 and COS-7 cells exhibited areceptor half-life of �3 h and thattreating cells with CDDO-Imgreatly reduced the rate of TGF�receptor degradation (Fig. 3A). Wealso carried out TGF� receptor deg-radation studies in mink lung epi-thelial cells, Mv1Lu cells, a cell linethat has been used to study TGF�signaling and degradation (12, 13).In Mv1Lu cells, the receptor half-life was observed to be �3 h (Fig.3A). Consistent with the resultsobserved with COS-7 and C2C12cells, the rate of TGF� receptor deg-radation was delayed to at least 8 hin the CDDO-Im-treated Mv1Lucells.A delay in receptor degradation

might be due to receptor exclusionfrom the compartment where deg-radation occurs or to a reduction inreceptor trafficking to that com-partment. To clarify this, we inves-tigated receptor internalization andco-localization with EEA1- or

caveolin-1-positive vesicles in the same cell. As we have pre-viously reported, Mv1Lu cells expressing HA-tagged T�RIIinternalize biotinylated TGF� into both EEA1- and caveolin-1-positive vesicles, as assessed by deconvolution immuno-fluorescence microscopy (15) (Fig. 3B, left). In the presenceof CDDO-Im, the amount of TGF� receptor trafficking wasreduced, since the majority of receptors after 1 h of incuba-tion at 37 °C did not accumulate in the perinuclear regions ofcells (Fig. 3B, right). However, co-localization with EEA1 orcaveolin-1-positive compartments, even in regions close tothe plasma membranes of cells, was still readily apparent(Fig. 3B, inset). Although this suggested that TGF� receptorshad indeed internalized from the plasma membrane, wewanted to test this further by carrying out acid washingexperiments (supplemental Fig. 2). Briefly, Mv1Lu cells sta-bly expressing T�RII were incubated at 4 °C with biotiny-

FIGURE 5. CDDO-Im delays TGF�-dependent cell migration but does not disrupt the association of TGF�receptors with Par6 protein. A, confluent monolayers of Rat2 fibroblasts were scratched and incubated at37 °C for 12 h in media supplemented with 0.2% fetal bovine serum (Control; top), media supplemented with0.2% fetal bovine serum plus 0.5 nM TGF� (middle), or media supplemented with 0.2% fetal bovine serum plus0.5 nM TGF� and 0.5 �M CDDO-Im (bottom). Cells were then fixed and imaged using bright field microscopy.The dotted lines indicate the starting point of cell migration. Representative micrographs from three experi-ments are shown. Bar, 0.1 mm. B, HEK293 cells were transiently transfected with cDNA encoding the proteinsindicated and incubated in the absence or presence of 1 �M CDDO-Im for 2 h prior to lysis and immunopre-cipitation (IP) with anti-Par6 antibodies. The immunoprecipitates were then subjected to SDS-PAGE and immu-noblotting with anti-HA or anti-FLAG antibodies to reveal protein complexes. One hundred micrograms oftotal protein lysates were immunoblotted (bottom) to assess protein expression. The bands corresponding toT�RI, T�RII, wild type Par6 (Par6 wt.), Par6 lacking the PB1 domain (Par6-�PB1), and IgG heavy chain (IgG HC) areindicated. Representative immunoblots from four experiments are shown.




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lated TGF� followed by streptavidin-Cy3. The cells werethen either acid-washed to remove any cell surface labelingor incubated at 37 °C in the absence or presence of 1 �MCDDO-Im for 1 h prior to incubation in acidic conditions.We observed that fluorescent probes bound to cell surfacereceptors at 4 °C were susceptible to acid washing and wereremoved. However, receptor-bound probes were notremoved by acidic treatment after the cells had been incu-bated at 37 °C for 1 h either in the presence or absence ofCDDO-Im (supplemental Fig. 2). This demonstrated thatalthough CDDO-Im altered TGF� receptor trafficking, it didnot inhibit receptor internalization from the plasmamembrane.Interestingly, we noted that caveolin-1-positive vesicles were

closer to the plasmamembrane in CDDO-Im-treated cells (Fig.3B). Moreover, co-localization of TGF� receptors with thecaveolin-1-positive compartment became quite prominent.The EEA1-positive vesicles also appeared to have an alteredsubcellular distribution in cells treated with CDDO-Im.To further assess the effect of CDDO-Im on the EEA1-posi-

tive endosomal compartment, we stained untreated or CDDO-Im-treated cells with anti-EEA1 antibodies and scored thenumber of cells that demonstrated a perinuclear staining pat-tern versus cells with a dispersed EEA1 staining pattern (Fig. 4).In cells that were not incubated with CDDO-Im, 72� 5% of thecells counted displayed EEA1 in a perinuclear distribution,whereas CDDO-Im-treated cells only had 27 � 4% perinuclearstaining (Fig. 4B). These data indicate that although CDDO-Imdoes not affect TGF� entrance into either the EEA1 or caveo-lin-1-positive compartments, it interferes with the dynamics oftheir trafficking. Furthermore, CDDO-Im leads to the dispersalof the EEA1 compartment and an accumulation of caveolin-1-positive structures in close proximity to the plasmamembranesof cells.

CDDO-Im Interferes with TGF�-dependent CellMigration—In orderto investigate a functional conse-quence of extending TGF�-dependent Smad signaling byCDDO-Im, we assessed TGF�-de-pendent cell migration, becauseCDDO-Im as well as the parentalCDDO compound exhibit potentantimetastatic activity in animalmodels (36). To assess polarized cellmovement, we carried out “woundhealing” assays using Rat2 fibro-blasts. In this assay, regions of con-fluent Rat2 cells were removed, andcell migration into the cell-freespace was assessed (Fig. 5A). Whencells were incubated in mediumcontaining low serum, the “wound”was observed even after 12 h ofincubation at 37 °C (Fig. 5A, top).The addition of TGF� to the mediacontaining low serum induced thecells at the edge of the scratch to

migrate perpendicularly into the cell-free space (Fig. 5A, mid-dle). Interestingly, the co-incubation of TGF� and CDDO-Iminhibited TGF�-dependent cell migration (Fig. 5A, bottom).

One possibility for this inhibition could be due to a disso-ciation of TGF� receptors from the polarity complex proteinPar6. This is based on the observation that the associationbetween TGF� receptors and Par6 is essential for EMT and cellmigration (2). We therefore carried out co-immunoprecipita-tion studies and observed that TGF� type I and II receptorsassociate with Par6 in untreated or CDDO-Im-treated cells(Fig. 5B). To assess the specificity of association, we used a Par6mutant that lacks the PB1 domain (Par6-�PB1). This domainwas previously shown to be essential for Par6-TGF� receptorassociation (34). As expected, we observed little associationbetween the Par6-�PB1 mutant and TGF� receptors (Fig. 5B).We next assessed the phosphorylation of Par6 by the TGF�type II receptor, using phosphoserine 345-specific antibodies.The phosphorylation of Ser-345 by TGF� type II receptors hasbeen shown to be necessary for TGF�-dependent EMT (34). Asseen in the co-immunoprecipitation studies, we did not observea change inTGF� receptor-dependent phosphorylation of Par6in the presence of CDDO-Im (data not shown).These results suggested that the inhibition of TGF�-de-

pendent migration by CDDO-Im was not dependent on theassociation of Par6 with TGF� receptors or its phosphoryl-ation and that other cellular targets could be part of theunderlying mechanism(s).CDDO-Im Alters Cytoskeletal Dynamics—The cellular

cytoskeleton regulates subcellular position of organelles, vesic-ular traffic, and cell polarity and cell migration (46, 47). Sincewe observed that CDDO-Im affects all of these processes invarious cell lines, we examined if the cytoskeletal network is apotential target for this compound. First, we evaluated the actincytoskeleton in control and CDDO-Im-treated cells by staining

FIGURE 6. CDDO-Im does not affect the actin cytoskeleton but alters the microtubule cytoskeleton.A, control Mv1Lu cells (top) or cells treated with 1 �M CDDO-Im (middle) or 10 �M cytochalasin B (bottom) for 1 hwere fixed and permeabilized. To visualize the actin cytoskeleton and the nuclei, cells were incubated withTexas Red (TR)-phalloidin (red) and DAPI stain (blue), respectively. Representative micrographs from threeexperiments are shown. Bar, 10 �m. B, Mv1Lu cells stably expressing the HA-tagged T�RII were incubated at4 °C with biotinylated TGF� followed by strepavidin-Cy3 (red; bTGF�) and transferred to 37 °C in the absence(Control) or presence of 1 �M CDDO-Im, 10 �M nocodazole, or 10 �M cytochalasin B. The cells were fixed,permeabilized, and probed with anti-�-tubulin antibodies (Tubulin; green). The nuclei were visualized usingDAPI staining (blue). The micrographs of the microtubule staining for the control (i) or the CDDO-Im-treatedcells (ii) were magnified to demonstrate microtubule structural differences (right). Representative micrographsfrom five experiments are shown. Bar, 10 �m.




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for filamentous actin (F-actin) using Texas Red-labeled phalloi-din (Fig. 6A, top and middle). In untreated cells, F-actin wasobserved as both a membrane-bound network, as well as stressfibers. In CDDO-Im-treated cells, the pattern of F-actin stain-ingwas indistinguishable from control cells, suggesting that theactin cytoskeleton is not a target of CDDO-Im. As a positivecontrol, we treated cells with cytochalasin B, which depolymer-

izes the actin cytoskeleton, andobserved marked structural differ-ences compared with control orCDDO-Im-treated cells (Fig. 6A,bottom).We next assessed howCDDO-Im

affected the microtubule network(Fig. 6B). Although CDDO-Im didnot cause depolymerization of themicrotubule network, the com-pound had a marked effect on theorganization and orientation ofmicrotubules. In control cells, themicrotubule network radiated out-ward from themicrotubule organiz-ing center toward the cell periphery(Fig. 6B, i). However, in CDDO-Im-treated cells, microtubules emanat-ing from the microtubule organiz-ing center clearly lacked thecharacteristic pattern of the micro-tubule network, appearing to snakethrough the cytoplasm in a lessorganized fashion (Fig. 6B, ii). Aspositive and negative controls, wetreated cells with nocodazole(which causes complete microtu-bule disruption) and cytochalasin B(which does not alter microtubulenetworking), respectively. As ex-pected, nocodazole but not cytocha-lasin B depolymerized microtu-bules and interfered with thetrafficking of TGF� receptors. Wealso examined TGF� receptorlocalization after CDDO-Im treat-ment and observed that the druginterfered with the distribution ofTGF� receptors. Thus, traffickingof TGF� receptors to the perinu-clear region is dependent onproper microtubule organizationand polarity. Together, these dataindicate that CDDO-Im alters themicrotubulenetwork in a fashion thatis distinct from the microtubule-de-polymerizing drug, nocodazole.To further investigate themecha-

nism of CDDO-Im-dependentmicrotubule network interference,we first investigated microtubule-

capping proteins, whichmodulatemembrane association of themicrotubule both at the plasma membrane and with vesicles(48). Clip-170 is a major capping protein that associates withthe plus-end of growing microtubules and links the microtu-bule to vesicles and to the plasma membrane via interactionswith IQGAP1 (32). Given the meandering nature of the micro-tubules in CDDO-Im-treated cells, we postulated that mem-

FIGURE 7. CDDO-Im induces Clip-170 to dissociate from microtubules. A, Rat2 fibroblasts transientlyexpressing GFP-Clip-170 were incubated in the absence (left) or presence of 1 �M CDDO-Im (right) for 1 h andwere then fixed, permeabilized, and immunostained with anti-�-tubulin antibodies (red). The co-localization ofGFP-Clip-170 (green) and microtubules (red) results in yellow staining. Bar, 10 �m. B, COS-7 cells were incubatedin the absence (Control) or presence of 1 �M CDDO-Im or 10 �M nocodazole (Nocod.) for 1 h and then subjectedto lysis at 37 °C to separate soluble proteins (S) from the cytoskeleton (C). Following processing for SDS-PAGE,cell lysates were immunoblotted with antibodies raised against Clip-170, IQGAP1, tubulin, actin, or Rac1. Therelative position of each resolved protein is indicated. Representative blots from four experiments are shown.C, Rat2 fibroblasts were microinjected with a GFP-Clip-170 cDNA and incubated for 4 h at 37 °C. The movementof GFP-tagged Clip-170 in control cells (top time course) or cells incubated with 0.1 �M CDDO-Im (bottom timecourse) were imaged using time lapse immunofluorescence microscopy. The starting point of movement of arepresentative Clip-170 cap is indicated by a red bar, and the white arrow follows the movement of the Clip-170signal along microtubules in the control cell and in large aggregates in the CDDO-Im-treated cell. Represent-ative images from three experiments are shown. Bar, 10 �m.




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brane and vesicular attachment might also be affected byCDDO-Im.To investigate the effect of CDDO-Im on Clip-170 associa-

tion with microtubules, we transiently transfected Rat2 fibro-blastswithGFP-taggedClip-170 and assessed its localization byimmunofluorescence microscopy. In control cells, Clip-170was observed to be associated with the positive end of growingmicrotubules (Fig. 7A, left), but in CDDO-Im-treated cells,Clip-170was redistributed to large puncta in the cytoplasm andwas no longer associated with microtubules (Fig. 7A, right). Tofurther address this, we examinedClip-170 associationwith thecytoskeleton by biochemical fractionation. For this, we solubi-lized membranes and separated the soluble fraction from theinsoluble cytoskeleton (Fig. 7B), a method that effectivelyremoves the majority of cellular organelles from thecytoskeleton fraction (supplemental Fig. 3). As expected,tubulin and most of actin protein was in the cytoskeletalfraction in control cells (Fig. 7B), whereas in nocodazole-treated cells, tubulin, but not actin, partitioned with the solublefraction. Consistent with the immunofluorescence studies,CDDO-Im of cells did not induce a solubilization and loss oftubulin from the cytoskeletal fraction. Interestingly, Clip-170was consistently present in the cytoskeletal fraction, regardlessof the treatment. This was surprising, because we assumed thatthe dissociation of Clip-170 from microtubules by eithernocodazole or CDDO-Im treatment would cause the majority

of the Clip-170 to be concentratedin the soluble fraction. However,Clip-170 remained in the cytoskel-etal fraction, suggesting that eitherCDDO-Im did not induce completeClip-170 dissociation from thecytoskeleton or that Clip-170 aggre-gated in punctate masses inresponse to CDDO-Im. In order todistinguish between the two possi-bilities, we visualized the dynamicsof Clip-170 mobility in real time bymicroinjecting cDNA encodingGFP-Clip-170 into Rat2 fibroblastsand carrying out time lapse immu-nofluorescence microscopy. In con-trol cells, the GFP-Clip-170 fol-lowed the growing microtubules(Fig. 7C and supplementalMovie 1).In the CDDO-Im treated cells, thepunctate aggregates that containedthe GFP-Clip-170 remained mobileat lower triterpenoid concentra-tions (0.1 �M; Fig. 7C and supple-mental Movie 2) but were immobi-lized at higher doses (1 �M;supplemental Movie 3), therebystalling the formation of a normalmicrotubule network. These dataindicate that CDDO-Im affects theorganization of the microtubularnetwork by interfering with the

function of the capping protein Clip-170.CDDO-Im Targets the Polarity Complex—In order to evalu-

ate the cellular target of triterpenoids, we attempted to identifythe subcellular localization of triterpenoids by utilizing a bio-tinylated version of CDDO. The biotinylated compound elicitsidentical cellular responses as the CDDO-Im, albeit at higherconcentrations (49), and allows for the assessment of triterpe-noid subcellular localization by immunofluorescence micros-copy (Fig. 8).As controls, biotin orCDDOdid not exhibit any fluorescence

signal (Fig. 8, A and B). However, we observed that CDDO-biotin localized in the nuclei and at the cell membrane inpatches that were consistent with the leading edge of migratingcells. To confirm this possibility, we immunostained scratchedRat2 fibroblasts using CDDO-biotin and Rac1 antibodies andobserved that Rac1 co-localized with biotinylated CDDO-bio-tin at the leading edge of the migrating cells (Fig. 8C).We next examined the subcellular localization of molecules

known to be involved in the leading edge of polarized cells (Fig.9). To carry this out, we first assessed the morphology of theleading edge of migrating Rat2 cells by DIC microscopy andobserved that untreated cells were elongated and had distinctlamellipodia, whereas CDDO-Im-treated cells were round. Inorder to assess this in a dynamic fashion, we carried out a realtime studywhere Rat2 fibroblasts werewounded and incubatedin control or CDDO-Im-containing media over time. Bright

FIGURE 8. Subcellular targeting of biotinylated CDDO. Rat2 fibroblasts were grown to 100% confluence andscratched to create a wound. After incubation for 6 h to allow cell polarization and migration, cells were fixed,permeabilized, and incubated with monoclonal anti-Rac1 antibodies (Rac1; green) and biotin (A), CDDO (B), orCDDO-biotin (C), followed by Cy2-labeled anti-mouse antibody and Cy3-labeled streptavidin. The co-localiza-tion of Rac1 (green) with CDDO-biotin (red) at the leading edge of migrating cells is demonstrated in the inset(yellow arrowheads). The blue arrow indicates the direction of cell movement, and DIC microscopy was includedto visualize the leading edge of migrating cells. Representative images from four experiments are shown. Bar,10 �m.




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field images were collected over 13 h and arranged in a movie(supplemental Movie 4).The positioning of CDDO-biotin at the leading edge of

migrating cells (Fig. 8C) and the CDDO-Im-dependent loss oflamellipodia (Fig. 9A, bottom) prompted us to investigate thelink between microtubules and the polarity complex, found atthe leading edge of migrating cells (Fig. 9B). For this line ofinvestigation, we carried wound-healing assays by first scratch-ingRat2 cells, allowing them to polarize andmigrate for 6 h, andthen incubated them in media containing CDDO-Im ornocodazole for an additional 2 h. This experimental approachwould allow us to study the effects of the drugs on the fate of the

polarity complex after it had beenpre-established for 6 h. We there-fore first assessed tubulin and Clip-170 in scratched Rat2 fibroblasts(Fig. 9C). In addition to the cellbody, control cells displayed endog-enous Clip-170 decorating the lead-ing edge of migrating cells, wheretubulin extended to the plasmamembrane. In contrast, CDDO-Imcaused endogenous Clip-170 to dis-perse from the leading edge intopunctate structures reminiscent ofthose observed after GFP-Clip-170expression (see Fig. 7A). We alsoexamined the localization of tubulinand Clip-170 proteins in nocoda-zole-treated cells. As expected,microtubules were disrupted, butdespite the accompanying alter-ation in cellular morphology, tubu-lin and Clip-170 staining remainedco-localized to the leading edge inthese cells (Fig. 9C, bottom). Thiswas notably different from theCDDO-Im treated cells, wherethere was an absence of Clip-170staining at the leading edge.The association of microtubules

to the leading edge of cells is facil-itated by Clip-170, which linksmicrotubules to IQGAP1 (32).Since Clip-170 failed to localize tothe leading edge of CDDO-Im-treated cells, we examined ifCDDO-Im also affects IQGAP1targeting to the leading edge ofpolarized cells. In untreated cells,IQGAP1 localized to the leadingedge of migrating cells, andCDDO-Im treatment abrogatedthis localization (Fig. 10A). More-over, general microtubule destabi-lization with nocodazole did notaffect IQGAP1 localization, con-sistent with our observation that

this drug does not affect the polarity complex once it hasalready been established (Fig. 10, A and B). Finally, in orderto determine if CDDO-Im causes a complete dissociation ofproteins present in the polarity complex, we assessed theassociation of IQGAP1 with Rac1 (Fig. 10C). We did not findany appreciable dissociation of Rac1 that was co-immuno-precipitated with IQGAP1 antibodies either in CDDO-Im-or nocodazole-treated cells. These data, in conjunction withthe observation that CDDO-Im induced the redistribution ofIQGAP1 at the plasma membrane, suggested that the local-ization of Rac1 and IQGAP1 to the leading edge might beaffected by triterpenoid treatment.

FIGURE 9. CDDO-Im interferes with cell morphology and the localization of Clip-170 at the leading edgeof migrating cells. A, Rat2 fibroblasts were grown to 100% confluence and scratched to create a wound. Thecells were then incubated for 4 h at 37 °C and treated with control medium or medium containing 1 �M

CDDO-Im for an additional 2 h before being fixed and processed for DIC microscopy. The dotted lines indicatethe starting point of cell migration. Representative micrographs from three experiments are shown. The bluearrows indicate the direction of cellular movement. Bar, 10 �m. B, model of microtubule association with asubset of polarity complex proteins at the leading edge of migrating cells. The microtubule capping protein,Clip-170, associates with IQGAP1. PKC� is shown in this model as a member of the polarity complex, and theblue arrow indicates the direction of cellular movement. C, Rat2 fibroblasts were grown to 100% confluence,scratched, and then incubated for 6 h at 37 °C. Cells were then incubated an additional 2 h in control medium(Control; top) or in media containing 1 �M CDDO-Im (middle) or 10 �M nocodazole (bottom) prior to fixation,permeabilization, and immunostaining with anti-Clip-170 (Clip-170) and anti-tubulin (Tubulin) antibodies. Thescratches were made in the horizontal plane above the cells shown, and the leading edges of migrating cellscontaining microtubule ends and Clip-170 are indicated with green and red arrowheads, respectively. Theyellow arrowheads show the co-localization of microtubule ends and Clip-170. The blue arrows indicate thedirection of cellular movement. Bar, 10 �m.




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To expand our analysis to other members of the polaritycomplex, we assessed the localization of Rac1 and PKC�, bothof which normally localize to the leading edge of cells undergo-ing polarized cell movement (Fig. 11). In untreated cells, bothRac1 and PKC�were observed to co-localize at the leading edgeof migrating cells (Fig. 11A). However, in response to CDDO-Im, we observed that although Rac1 localized to themembrane,it was less organized and dispersed along the cell membrane.Furthermore, PKC� stainingwas altogether absent from the cellmembrane of CDDO-Im-treated cells (Fig. 11B). Nocodazoletreatment altered the morphology of leading edge cells, andRac1 was now found in numerous protrusive structures allaround the cell (Fig. 11C). In these cells, PKC� remained co-localized with Rac1, consistent with the lack of change inIQGAP1 or Clip-170 localization observed after nocodazoletreatment. These results indicate that the function and assem-bly of the polarity complex is disrupted in response toCDDO-Im treatment and that this effect is not dependent ongeneralmicrotubule disruption, since polarity complex constit-uents remained associated in nocodazole-treated cells.Based on our observations that CDDO-Im does not disrupt

the association between Par6 and TGF� receptors (Fig. 5B) but

does disrupt the localization ofmembers of the polarity complex(Figs. 9–11), we predicted that thelocalization of TGF� receptors andPar6 at the leading edge might bereduced and/or abrogated in thepresence of CDDO-Im. To test this,we assessed the localization of Par6and T�RII by immunofluorescencemicroscopy (Fig. 12). In untreatedcells, we observed that both Par6and TGF� receptors were indeedpresent at the leading edge ofmigrating cells and that they bothco-localized with Rac1 (Fig. 12).However, in response to CDDO-Imtreatment, the amounts of Par6 andT�RII at the leading edge of cellswere markedly reduced (Fig. 12).These results further support ourobservations that CDDO-Im dis-rupts TGF�-dependent cell migra-tion by disrupting the polaritycomplex.Taken together, our results

demonstrate that the triterpenoidCDDO-Im alters TGF� receptortrafficking and signal transduc-tion, microtubule-plasma mem-brane attachments, and vesiculartransport. The mechanism ofaffecting cell polarity and migrationis dependent on the disruption ofClip-170 capping of microtubulesand disrupting microtubule attach-ments with the polarity complex.

Furthermore, the co-localization of triterpenoid with Rac-1at the leading edge of migrating cells positions it to interferewith cell polarity by disrupting the localization of IQGAP1,PKC�, Par6, and TGF� receptors at the leading edge ofmigrating cells.

DISCUSSION

TGF� is a growth factor that acts as a tumor suppressor orpromoter, depending on cellular context (4, 50). TGF� recep-tors propagate several signaling pathways, two of which areessential for EMT: the Smad pathway (51, 52) and the Par6pathway (34, 53). In this study, we found that CDDO-Im inhib-its TGF�-dependent cell migration. In order to identify themechanism, we first assessed its effect on TGF� signaling. Aspreviously described in studies using U937 and HL60 cells (37,39), we found that CDDO-Im extended the phosphorylationprofile of Smad2; however, further investigation suggested thatthis was not due a decrease in Smad2 phosphatase activity (Figs.1 and 2 and supplemental Fig. 1). We next assessed receptortrafficking and degradation. Previous studies indicated that byperturbing the lipid raft compartment, the distribution of inter-nalized cell surface TGF� receptors could be shifted from the

FIGURE 10. Reduction of IQGAP1 localization at the leading edge of migrating cells in response toCDDO-Im treatment. A, cells were scratched and allowed to migrate into the wound for 6 h in order toestablish cell polarity prior to incubation with control medium (top) or media containing 1 �M CDDO-Im(middle) or 10 �M nocodazole (bottom) for an additional 2 h. Cells were then fixed, permeabilized, and immu-nostained for endogenous Rac1 (green) and IQGAP1 (red) protein. A representative area of interest (white box)from each condition was enlarged and shown (inset). The green and red arrows indicate Rac1 and IQGAP1 at theleading edge of migrating cells, respectively. The co-localization of Rac1 with IQGAP1 is indicated by yellowarrowheads. Bar, 10 �m. B, quantitation of cells containing Rac1 or IQGAP1 at the leading edge of migratingcells was carried out as described under “Experimental Procedures” and graphed (n � 3 � S.D.). C, untreatedcells (Control) or cells incubated with either CDDO-Im or nocodazole (Nocod.) were lysed and immunoprecipi-tated with nonimmune IgG (Non-IM IgG) or anti-IQGAP1 antibodies (�-IQGAP1) and immunoblotted (Blot) withanti-IQGAP1 or anti-Rac1 antibodies. The relative mobilities of Rac1 or IQGAP1 are indicated on the left of eachpanel. One hundred micrograms of total protein lysates were immunoblotted and shown on the left. Repre-sentative blots from three experiments are shown.




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caveolin-1-positive compartmentto the EEA1 early endosomal com-partment, leading to enhanced sig-naling and delayed receptor degra-dation (15). We observed thatCDDO-Im did delay receptor deg-radation; however, unlike lipid raft-destabilizing agents, such as nysta-tin, CDDO-Imdid not shift receptorequilibrium toward the caveolin-1-positive compartment. Instead,CDDO-Im delayed overall receptortraffic and induced both caveolin-1-and EEA1-positive vesicles to belocalized to the peri-plasma mem-brane regions of cells (Figs. 3 and 4).This led us to conclude that thecompound was affecting a cellularstructural component and quitepossibly the cytoskeleton, sinceboth the actin and microtubulecytoskeleton have been shown tobe important for the trafficking ofvesicles from the plasmamembraneto the cell interior (21, 46, 54, 55).Further study revealed that

although the actin cytoskeleton wasnot morphologically affected, themicrotubule network became disor-ganized (Fig. 6). The disorganiza-tion of vesicular positioning in thecell is a hallmark of microtubulecatastrophe brought on by destabi-lizing drugs, such as nocodazole(20). Indeed, the microtubule net-work will disperse if cells are treatedwith high triterpenoid concentra-tions (56). However, in the presentstudy,we used lower concentrationsof CDDO-Im, consistent with theconcentrations used in animal stud-ies (57–59), and found thatCDDO-Im does not dissolve themicrotubule cytoskeleton. Rather,we observed that microtubules inCDDO-Im-treated cells take on aloopingmorphology that is reminis-cent of a lack of proper attachmentto the cell membrane and loss of cellpolarity. We therefore tested ifmicrotubule-capping proteins mightbe affected, since they act as anchor-age points both between microtu-bules and vesicles and betweenmicrotubules and the cell mem-brane (26, 48). The latter processinvolves a number of intermediatemolecules, such as Clip-170, and

FIGURE 11. CDDO-Im disrupts PKC� localization at the leading edge of polarized cells. Rat2 fibroblastmonolayers were scratched and allowed to grow into the wound for 6 h in order to polarize before beingincubated in the absence (Control; A) or presence of 1 �M CDDO-Im (B) or 10 �M nocodazole (C) for anadditional 2 h. Cells were then fixed, permeabilized, and immunostained for endogenous Rac1 (green) orPKC� (red), using anti-Rac1 and anti-PKC� antibodies, respectively. A representative area of interest (whitebox) from each condition was enlarged and shown (inset). The green and red arrows indicate Rac1 and PKC�at the leading edge of migrating cells, respectively. The co-localization of Rac1 with PKC� is indicated byyellow arrowheads. The blue arrows indicate the direction of movement. Representative images from fourexperiments are shown. Bar, 10 �m.




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members of the polarity complex, Cdc42-Rac1, via IQGAP1(32, 33, 48).Interestingly, Clip-170 and IQGAP1were found to dissociate

from the leading edge of cells in response to CDDO-Im. Theseobservations explain both the loss of vesicular traffic to the cellinterior and the loss of microtubule targeting and associa-tion with the leading edge of cells (Figs. 7, 9, and 10). Of note,nocodazole, a drug that disrupts microtubules, was unable toaffect the polarity complex after the cells were allowed topolarize in the absence of drugs. These observations indicatethat the effects of CDDO-Im on Clip-170 dissociation frommicrotubules and the loss of concentration of molecules inthe polarity complex are distinct from compounds that dis-sociate Clip-170 from microtubules via microtubule catas-trophe. Our results also suggest that the association of dif-ferent members of the polarity complex have variablestability. Indeed, we found that Rac1 remains associated withIQGAP1 regardless of the pharmacological treatment (Fig.10). This stable association is also reflected in the fraction-ation studies, since the majority of Rac1 and IQGAP1 bothpartitioned with the soluble fractions, whereas Clip-170 wasconcentrated in the cytoskeletal fractions (Fig. 7). However,the dissociation of Clip-170 from microtubules and the lossof PKC� from the polarity complex were exquisitely sensitiveto CDDO-Im treatment. This has implications not onlyfor TGF�-dependent cell migration but for migration de-

pendent on other growth factorsand receptors as well. Indeed,CDDO-Im inhibits serum-stimu-lated Rat2 fibroblast migration(supplemental Movie 4). However,in the case of TGF�-dependent cellmigration, our results indicate thatthe location of Par6 and TGF�receptors may be important.It was interesting that TGF�

receptors remained associated withPar6 even as TGF�-dependentmigrationwas abrogated byCDDO-Im. When we assessed the associa-tion of Par6 with TGF� receptors,we detected no difference in theassociation or phosphorylation ofPar6 in response to CDDO-Imtreatment. Therefore, this aspect ofTGF�-dependent migration wasnot perturbed. The localization ofthe triterpenoid to the leading edgeof migrating cells was intriguing(Fig. 8), since it may suggest that themechanism of CDDO-Im blockcould be the modulation of proteinsat this locus. We reasoned that per-haps the loss of IQGAP1, PKC�, andRac1 at the leading edge of migrat-ing cells would be accompaniedwith a loss of Par6 and TGF� recep-tors. This was confirmed when we

assessed Par6 and TGF� receptor localization by immunofluo-rescence microscopy and found both partners to be greatlyreduced at this site (Fig. 12). Further investigation of howCDDO-Immodulates TGF� receptor signaling will be interest-ing, since the concentration of TGF� receptors in various sub-cellular locations has been shown to be essential to stimulateSmad2 phosphorylation on endosomal membranes (8, 9, 15,42–45), EMT at tight junctions (34), and degradation of RhoAin lamellipodia and filopodia (60–62).Finally, our results may also give some insight into themech-

anism of the antimetastatic and antiproliferative effects ofCDDO-Im in animal studies (57–59) and would be an interest-ing area of study particularly for understanding mechanisms ofcell migration and metastasis in human cancer. A general classof antimetastatic agents, microtubule-destabilizing drugs,affect cell motility, migration, and metastasis (63). Therefore,combining anti-microtubule drugs with drugs such asCDDO-Im that target the attachment sites between microtu-bules and the cell membrane may be an effective therapeuticapproach to metastasis.

Acknowledgments—We thank the members of the Wrana laboratoryfor advice and support, Kavitha Sengodan for technical assistance,and Boun Thai for technical assistance and critical evaluation of themanuscript.

FIGURE 12. CDDO-Im disrupts Par6 and TGF� receptor localization at the leading edge of migrating cells.Rat2 fibroblast monolayers were scratched and allowed to grow into the wound for 6 h before being incubatedin the absence (Control), or presence of 1 �M CDDO-Im for an additional 2 h. Cells were then fixed, permeabi-lized, and immunostained for endogenous Rac1 (green) and Par6 (red) using anti-Rac1 and anti-Par6 antibodies(A) or for endogenous Rac1 (green) and T�RII (red) using anti-Rac1 and anti-T�RII antibodies, respectively (B). Arepresentative area of interest (white box) from each condition was enlarged and shown (inset). The greenarrowheads indicate Rac1, and red arrowheads indicate Par6 or T�RII at the leading edge of migrating cells. Theco-localization of Rac1 with Par6 or T�RII is indicated by yellow arrowheads. The blue arrows indicate thedirection of movement. Bar, 10 �m. Cells containing Rac1, Par6, or T�RII at the leading edge were quantitatedfrom three experiments carried out as described in A and B � S.D. and graphed (C).




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B. Sporn, Jeffrey L. Wrana and Gianni M. Di GuglielmoCiric To, Sarang Kulkarni, Tony Pawson, Tadashi Honda, Gordon W. Gribble, Michael

Cell Migration by Affecting the Cytoskeleton and the Polarity Complex-dependent Signaling andβAcid-Imidazolide Alters Transforming Growth Factor

The Synthetic Triterpenoid 2-Cyano-3,12-dioxooleana-1,9-dien-28-oic

doi: 10.1074/jbc.M704064200 originally published online February 18, 20082008, 283:11700-11713.J. Biol. Chem.

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