Patrick Fafet

Cosette Rebouissou

Thierry Maudelonde

Marie-Luce Vignais

Opposite Effects of Transforming Growth Factor-␤ Activation and Rho-Associated Kinase Inhibition on Human Trophoblast Migration in a Reconstituted Placental-Endometrial Coculture System

Keywords: EVCT, Extravillous cytotrophoblastic cell, FBS, fetal bovine serum, HLA, human leukocyte antigen, IGFBP, IGF binding protein

Placental implantation involves highly regulated trophoblast invasion of the endometrial stroma. TGF␤ is a known regulator of this process. This study examines the effect of TGF␤ on extravillous cytotrophoblastic cell (EVCT) migration in cocultures of first-trimester human chorionic villus explants and primary human endometrial fibroblasts. Migration of EVCTs was followed by phase-contrast time-lapse microscopy and was shown to highly depend on the endometrial fibroblast matrix. Interstitial EVCT invasion was also analyzed by confocal microscopy of fluorescently prelabeled trophoblasts and endometrial fibroblasts. As expected, addition of TGF␤ led to inhibition of EVCT invasion of the endometrial cell layer. This inhibition was characterized by formation of compact EVCT stacks at migration fronts and displacement of endometrial fibroblasts. We tested the role of the RhoA/Rho-associated kinase (ROCK) pathway, a TGF␤-dependent pathway known to regulate cell migration. Interestingly, blocking ROCK with the chemical inhibitor Y27632 had an effect opposite to TGF␤ activation because it promoted superficial EVCT migration on the endometrial cell layer. These data suggest a role for ROCK in the TGF␤-dependent control of trophoblast migration. Furthermore, they indicate that even though ROCK signaling plays a role in human trophoblast cell invasion, EVCT migration can still occur in the absence of ROCK activity.

P LACENTAL IMPLANTATION during pregnancy gives rise to naturally occurring cell invasion. In humans, trophoblast cells produced from the trophectoderm of the blastocyst are responsible for the invasion of the entire endometrium and a fraction of the myometrium in the so-called interstitial invasion [START_REF] Norwitz | Implantation and the survival of early pregnancy[END_REF]. During placental implantation a fraction of the chorionic villi, called the anchoring villi, develop and produce multilayered columns of nonpolarized extravillous cytotrophoblastic cells (EVCTs). These cells further differentiate into highly invasive EVCTs that migrate as individual cells and are involved in the interstitial invasion of the endometrial decidua and myometrium [START_REF] Bischof | Irminger-Finger I 2005 The human cytotrophoblastic cell, a mononuclear chameleon[END_REF].

Trophoblast and cancer cells share striking similarities in their migratory and invasive capacities, which suggests the use of common molecular mechanisms. Cancer cells are known to actively interact with the surrounding stroma that can either act as a powerful tumor suppressor or, alternatively, help promote tumor growth and invasion [START_REF] Bhowmick | Tumor-stroma interactions[END_REF][START_REF] Bissell | Context, tissue plasticity, and cancer: are tumor stem cells also regulated by the microenvironment?[END_REF]. Similarly, successful trophoblast invasion and blastocyst implantation depend on widespread metabolic and endocrine changes of the endometrium, a process that is known as endometrial decidualization and that is under the control of the trophoblast itself. Decidualized endometrial fibroblasts express a wide array of cytokines and growth factors such as prolactin and IGF binding protein (IGFBP)-1 that are established markers of decidualization and can regulate EVCT migration [START_REF] Irving | Functional role of cell surface integrins on human trophoblast cell migration: regulation by TGF-␤, IGF-II, and IGFBP-1[END_REF][START_REF] Brar | Gene induction and categorical reprogramming during in vitro human endometrial fibroblast decidualization[END_REF]. In vitro, decidualization of endometrial fibroblasts can be achieved by the combined action of progesterone and cAMP [START_REF] Tang | Cyclic adenosine 3Ј,5Ј-monophosphate induces prolactin expression in stromal cells isolated from human proliferative endometrium[END_REF]. It can also be induced ex vivo by the interaction with first-trimester trophoblast explants as shown recently by Popovici et al. [START_REF] Popovici | Gene expression profiling of human endometrial-trophoblast interaction in a coculture model[END_REF].

Apart from their marked similarities, migration and tissue invasion by trophoblast and cancer cells also bear major differences. Most notably, contrary to malignant cells, extravillous cytotrophoblastic cells display a controlled invasive capacity that is limited in both space and time. Loss of this control can lead to pathological conditions such as development of hydatidiform moles or choriocarcinomas. This indicates that, despite comparable invasive characteristics, trophoblast and cancer cell behaviors rely on clearly distinct regulatory circuits.

TGF␤ is a pluripotent cytokine that plays key roles in numerous cellular processes [START_REF] Derynck | Differentiation plasticity regulated by TGF-␤ family proteins in development and disease[END_REF]. Functions such as proliferation or differentiation can be regulated positively or negatively, depending on cell type and context [START_REF] Byfield | Lateral signaling enhances TGF-␤ response complexity[END_REF][START_REF] Schmierer | TGF␤-SMAD signal transduction: molecular specificity and functional flexibility[END_REF]. Because of its negative effect on epithelial cell proliferation, TGF␤ signaling was proposed as a tumor suppressor pathway. However, TGF␤ overexpression by cancer cells, loss of TGF␤-dependent control of proliferation in these cells and otherwise TGF␤-induced metastatic invasiveness now make TGF␤ signaling a target for cancer therapy [START_REF] Pennison | Targeting transforming growth factor-␤ signaling[END_REF].

TGF␤ is closely associated with tissue remodeling in the physiological processes of embryonic development and placental implantation. It also regulates invasion of the endometrial decidua by trophoblast [START_REF] Irving | Functional role of cell surface integrins on human trophoblast cell migration: regulation by TGF-␤, IGF-II, and IGFBP-1[END_REF][START_REF] Graham | Mechanism of control of trophoblast invasion in situ[END_REF][START_REF] Godkin | Transforming growth factor ␤ and the endometrium[END_REF][START_REF] Rama | TGF␤1 induces multiple independent signals to regulate human trophoblastic differentiation: mechanistic insights[END_REF][START_REF] Karmakar | Modulation of ezrin and E-cadherin expression by IL-1␤ and TGF-␤1 in human trophoblasts[END_REF](17)[START_REF] Jones | TGF-␤ superfamily expression and actions in the endometrium and placenta[END_REF]. TGF␤ and its receptors are expressed by both the placenta and endometrium (14, 19 -22). After degradation of the endometrial epithelium and in sharp contrast to its effect on cancer cells, TGF␤ acts as a major repressor of cytotrophoblast invasion and migration [START_REF] Irving | Functional role of cell surface integrins on human trophoblast cell migration: regulation by TGF-␤, IGF-II, and IGFBP-1[END_REF][START_REF] Graham | Mechanism of control of trophoblast invasion in situ[END_REF][START_REF] Jones | TGF-␤ superfamily expression and actions in the endometrium and placenta[END_REF]. Interestingly, this TGF␤-dependent inhibition of trophoblast invasion is lost in malignant choriocarcinomas, such as JARs or JEGs [START_REF] Graham | Resistance of malignant trophoblast cells to both the anti-proliferative and anti-invasive effects of transforming growth factor-␤[END_REF].

Various signaling pathways mediate the biological effects of TGF␤. Among these, the pathway involving the small GTPase RhoA and its downstream effector Rho-associated kinase (ROCK) has been associated with cell migration regulation [START_REF] Raftopoulou | Cell migration: Rho GTPases lead the way[END_REF][START_REF] Bierie | Tumour microenvironment: TGF␤: the molecular Jekyll and Hyde of cancer[END_REF]. ROCK is reported to increase the invasive capacity of tumor cells, a capacity blocked by the specific Y27632 inhibitor [START_REF] Itoh | An essential part for Rho-associated kinase in the transcellular invasion of tumor cells[END_REF][START_REF] Sahai | Mechanisms of cancer cell invasion[END_REF]. Likewise, a function for the RhoA-ROCK signaling cascade was also proposed for the regulation of extravillous cytotrophoblast migration (28 -30).

Several model systems, which rely on different sources of trophoblast and matrix, have been used to study trophoblast migration and invasion ex vivo. A number of trophoblast cell lines derived from choriocarcinomas or artificially immortalized from normal placentas have been described and proven powerful research tools [START_REF] King | Cell culture models of trophoblast II: trophoblast cell lines-a workshop report[END_REF]. However, because of the diverse origins and means of immortalization of these cell lines, their characteristics can widely vary as recently shown by comparative microarray analysis of the BeWo and JEG3 cell lines [START_REF] Burleigh | Microarray analysis of BeWo and JEG3 trophoblast cell lines: identification of differentially expressed transcripts[END_REF]. Alternatively, trophoblast cells or chorionic explants freshly isolated from first-trimester or term placentas can be used, the explants offering the valuable advantage of maintaining an intact villus structure most likely capable of secreting diffusible regulatory factors (17,[START_REF] Vicovac | Trophoblast differentiation during formation of anchoring villi in a model of the early human placenta in vitro[END_REF][START_REF] Bellingard | Identification of prometalloproteinase-3 as a major protein secreted by human endometrial fibroblasts and inhibited by coculture with trophoblast cells[END_REF][START_REF] Aplin | Development of cytotrophoblast columns from explanted first-trimester human placental villi: role of fibronectin and integrin ␣5␤1[END_REF]. Artificial matrices like collagen or Matrigel have been used to support trophoblast migration (17,[START_REF] Aplin | Development of cytotrophoblast columns from explanted first-trimester human placental villi: role of fibronectin and integrin ␣5␤1[END_REF][START_REF] Fournier | Involvement of PPAR␥ in human trophoblast invasion[END_REF]. However, a coculture of the tissues that interact in vivo is more likely to reproduce naturally occurring interactions as it was recently shown for the characterization of trophoblastic endovascular invasion [START_REF] Cartwright | Trophoblast invasion of spiral arteries: a novel in vitro model[END_REF]. To study trophoblastic interstitial invasion, cocultures between endometrial tissues and placental villous explants or between cells isolated from these tissues have been used [START_REF] Vicovac | Trophoblast differentiation during formation of anchoring villi in a model of the early human placenta in vitro[END_REF][START_REF] Bellingard | Identification of prometalloproteinase-3 as a major protein secreted by human endometrial fibroblasts and inhibited by coculture with trophoblast cells[END_REF]. Recently gene expression profiling of human endometrial stromal cells grown in coculture with first-trimester trophoblast explants showed the impact of trophoblast on endometrial gene expression therefore validating this experimental approach [START_REF] Popovici | Gene expression profiling of human endometrial-trophoblast interaction in a coculture model[END_REF].

Whereas there is strong evidence that TGF␤ regulates trophoblast invasion, little is known about the signaling pathways involved. It is also unclear how this regulation takes place and what mechanisms are involved. The aim of the present investigation was to characterize the effect of TGF␤ on the regulation of interstitial migration/invasion of EVCTs and investigate the role of the RhoA/ROCK pathway. To address these questions, we set up an in vitro coculture system between human trophoblastic villi and primary endometrial fibroblasts. This system enables trophoblast-dependent decidualization of the endometrium and therefore recapitulates some of the interactions taking place in vivo between these two tissues. We characterized the migration of extravillous trophoblast cells and confirmed the inhibitory effect of TGF␤ on trophoblast migration. Furthermore, we observed that inhibition of ROCK by Y27632 treatment induced an effect opposite to that of TGF␤ and unexpectedly promoted two-dimensional migration of trophoblast cells.

Materials and Methods

Patients and samples

Endometrial tissues were obtained from women undergoing cesarean sections at the Hospital Arnaud de Villeneuve (Montpellier, France). They were at term for a normal pregnancy. Trophoblast villi were isolated from placentas (n ϭ 20) obtained from legal early pregnancy terminations of uncomplicated, unwanted pregnancies. Abortions were induced by mifepristone. All samples were obtained at 4 -7 wk gestational age. Pregnancy was dated by echography and measurement of both the crown-rump length and mean gestational sac diameter. Average mother age was 27 Ϯ 8 yr. All samples were obtained after informed written consent of the patients and the protocol was approved by the local ethical committee.

Endometrial fibroblast isolation and in vitro decidualization

Endometrial samples were obtained from normal pregnant women (n ϭ 4) undergoing cesarean section at term. Two-millimeter-long explants were mechanically chopped from fresh endometrial tissue and seeded on 6-cm dishes in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS). These endometrial fibroblasts had a doubling time of roughly 24 h and entered senescence after about 50 d in culture. They were passaged twice a week and used before passage 6. For in vitro decidualization, endometrial fibroblasts were plated in FBS 10% at a concentration of 10 5 cells per 4-cm dish. When confluent, they were cultured in the presence of medroxyprogesterone acetate (1 m) and 8-bromoadenosine-cAMP (0.5 mm; Sigma, Saint-Quentin Fallavier, France) for 4 d.

Establishment of cocultures

After uterine evacuation, placentas were immediately collected in DMEM supplemented with heat-inactivated FBS 10%, penicillin (200 U/ml), streptomycin (200 g/ml), fluconazole (4 g/ml; Pfizer, Sandwich, UK), and amphotericin B (20 g/ml; Bristol-Myers Squibb, Princeton, NJ; DMEM). They were processed for dissection within the next 2 h. Meanwhile, they were kept at room temperature and protected from light. Before dissection, placentas were gently washed with warm PBS three times or more to eliminate blood. Placentas were then incubated for 20 -30 min in the above-mentioned DMEM. Placental villosities were dissected with sterile tweezers and scissors to get tissue pieces about 1 mm long. Dissociation of villosities in small pieces was done by pipetting them up and down with a 10-ml pipette. Villosities were then transferred to a 50-ml Falcon tube and let to decant. They were rinsed once and resuspended in a medium composed of 50% of the above-mentioned DMEM and 50% of Ham F12 medium supplemented with CaCl 2 (2 mm), MgSO 4 (2 mm), and NaHCO 3 (0.5 mm; DMEM/Ham medium).

Endometrial fibroblasts were seeded on glass coverslips (10 5 cells in 4 cm diameter dishes) in DMEM supplemented with FBS 10% 3 d before the coculture so that fibroblasts were confluent by the time coculture was started. Before setting up the coculture, the fibroblast medium was changed to DMEM/Ham medium. Approximately 10 trophoblast explants were seeded per 4-cm dish and let to adhere for 16 h at 37 C. At that point, the medium was aspirated, and plates were rinsed once with the DMEM/Ham medium to eliminate all unattached villosities and further incubated in the same medium. This was considered to be time zero for all measures of trophoblast migration and for further treatment.

Treatments

TGF␤1 (R&D Systems, Minneapolis, MN) was used at a concentration of 10 ng/ml. The pharmacochemical inhibitor Y-27632 (Alexis Biochemi-cals, Lausen, Switzerland) was used at a final concentration of 5-10 m. The pan anti-TGF␤ antibody is a monoclonal murine antibody (clone 1D11; R&D Systems) used at a concentration of 4 g/ml. Treatments were done at 37 C from 8 to 24 h as indicated.

Immunofluorescence

Cells were fixed with paraformaldehyde (4%) for 10 min and permeabilized with Triton (0.1%, 2 min). Alternatively, for IGFBP1 and prolactin (PRL) labeling, cells were permeabilized with ethanol (EtOH 70%, 30 min at 4 C). After three PBS washes, saturation was performed with FBS 10% for 30 min at room temperature. Cells were incubated for 1 h at room temperature with the primary antibodies in 150 l PBS/ BSA 1%. The antibodies used were the following: mouse antihuman cytokeratin 7 (1:400, clone OV-TL 12/30; DakoCytomation, Glostrup, Denmark), mouse antivimentin cy3 conjugate (clone V9, 1:2000; Sigma), mouse anti-human leukocyte antigen (HLA)-G (clone 4H84, 1:100, Exbio, Vestec, Czech Republic), rabbit anti-PRL (1:400) and anti-IGFBP1 (1:200; Upstate Biotechnology, Lake Placid, NY). Immunofluorescence was monitored by incubation at room temperature for 1 h with either Cy-3 or fluorescein isothiocyanate-conjugated antirat, antimouse, or antirabbit IgG. Hoechst 33342 (Molecular Probes, Leiden, The Netherlands) was used for nuclei staining. After three additional PBS washes, slides were mounted with ProLong Gold antifade reagent (Molecular Probes). For vital dye labeling, the green 5-chloromethyl-fluorescein diacetate and red CMTPX CellTracker probes (Molecular Probes) were used. Briefly, confluent endometrial fibroblasts were incubated for 30 min at room temperature in DMEM supplemented with FBS 10% and the green 5-chloromethyl-fluorescein diacetate (4 m). The 1-mm-long trophoblastic villi were incubated for 45 min at room temperature in DMEM supplemented with FBS 10% and the red CMTPX (10 m). Cells and villosities were then independently rinsed twice with FBS 10% and further incubated with FBS 10% at 37 C for 30 min. They were rinsed twice more with FBS 10% and put in the DMEM/Ham medium (see above). Cocultures were then set up between green-stained fibroblasts and red-stained villi. The reverse combination of CellTracker probes for, respectively, the endometrial fibroblasts and the trophoblastic villi was also used.

Imaging

Trophoblast cell invasion was followed by time lapse with a phasecontrast DMIR microscope (Leica, Heidelberg, Germany) in an incubation chamber providing controlled temperature, CO 2 concentration, and hygrometry. Pictures were taken every 2 min during 24 -48 h using a ORCA 100 and the HPDCPx32 program (Hamamatsu, Bridgewater, NJ). After imaging, all time points were compiled and exported as a Quicktime (avi) file using the MetaMorph software (Universal Imaging Corp., West Chester, PA). Alternatively, images were taken with a Leica DMIRE 2 2002 microscope and a MicroMax 1300 Y/HS (1999) camera (RS-Princeton Instruments, Trenton, NJ) with the MetaMorph 7 acquisition program. Image analysis and measurement of migration velocities was performed with the MetaMorph and Excel (Microsof Corp., Redmond, WA) softwares. Individual cell tracking was done with the Flash MX 2004 (Macromedia, San Francisco, CA) software. Immunofluorescence and CellTracker stainings were visualized with a LSM 510 Meta confocal laser system (Carl Zeiss, Jena, Germany). Images were converted with MetaMorph and processed with the Adobe Photoshop software (Adobe Systems Inc., San Jose, CA).

Statistical analysis of phase-contrast images

For analysis of EVCT migration, two-way ANOVA was used to compare the effects of the culture treatments (TGF␤1, Y27632, or control) and the different placental origins. Unpaired Student's t tests with Welch correction were performed to compare villi distributions for the different experimental conditions. Data on lacune formation after TGF␤1, Y27632, or control treatments were compared as nonnormal distributions using the Mann-Whitney rank sum test. Statistical analysis was done with the Prism software (GraphPad Inc., San Diego, CA) (*, P Ͻ 0.05; **, P Ͻ 0.01; ns, not significant).

Results

Reconstitution of the trophoblastic invasion of the endometrium

For setting up our ex vivo coculture system involving placental explants and endometrial fibroblasts, we used placentas from early elective pregnancy terminations (4 -7 wk gestation). Within a couple of hours of placenta expulsion, trophoblast villi were trimmed off the placentas, cut into 1to 2-mm pieces and seeded on top of confluent layers of primary endometrial fibroblasts. The human primary endometrial fibroblasts were isolated independently from endometrial tissues removed during cesarian section. They doubled every 24 h and entered senescence after about 50 generations (data not shown). They were used before passage 6. For setting up cocultures of placental explants and fibroblasts, trophoblast villi were let to adhere to the fibroblast layer for 12-16 h at 37 C. At that time, culture dishes were gently washed with culture medium to get rid of any unbound placental material and cultures were submitted to various treatments (see following text) or left untreated (Figs. 1 and2). Production of trophoblast cells by the villi and migration of these EVCTs away from the villi, onto the fibroblast layer, were followed by phase-contrast time-lapse analysis (Fig. 1). Important changes in trophoblast cell shapes were observed, suggesting an adaptation of the EVCTs to the fibroblast extracellular matrix and their ability to squeeze through narrow spaces. Trajectories of isolated trophoblastic cells were tracked and confirmed that: 1) cells migrate away from the villosity, 2) EVCT trajectories are not strictly straight and show a strong dependence on fibroblast orientation patterns, and 3) consequently, EVCT migration velocities for a given trophoblastic cell also depend on the fibroblast cell layer pattern, ranging in this case from 10 to 40 m/h (see trajectories I, II, and III in Fig. 1B).

To follow EVCT migration over longer distances, we performed phase-contrast time-lapse acquisition of contiguous fields followed by image reconstruction over a 35-h period (see reconstituted 5 ϫ 5 contiguous fields in Fig. 2). Velocities of trophoblast cell migration fronts were determined (see migration directions I, II, and III in Fig. 2B). As for individual trophoblast cells, the measured velocities showed a clear dependence on fibroblast orientation patterns with a faster EVCT migration rate along the longer fibroblast axis. From a total number of six placentas analyzed, and 30 EVCT speed values determined, we found that EVCT migration velocities in our coculture system ranged from 11 to 46 m/h, with an average EVCT migration velocity of 23 m/h (data not shown).

Trophoblast-induced decidualization of the endometrial fibroblasts

The endometrial fibroblasts were characterized by vimentin expression and their cAMP and progesterone-induced decidualization, as shown by the expression of specific markers of decidualization. Indeed, expression of PRL and IF-GBP1 was detected under these decidualizing conditions but not without treatment (control conditions) (Fig. 3A).

To assess how representative of in vivo conditions our model culture system was, we investigated whether endo- metrial decidualization, known to take place in vivo in response to trophoblast invasion, was also taking place in our coculture system. We detected expression of PRL in endometrial fibroblasts cocultured with trophoblastic villi (Fig. 3B). Similar data were obtained in independent experiments using two distinct placentas. This indicated that endometrial fibroblasts in culture with trophoblastic villi could undergo decidualization, without addition of decidualization-inducing reagents such as cAMP or progesterone (see above). These data showed that an effective interaction between the two cell types, respectively, the trophoblastic epithelial cells and the endometrial fibroblasts, was taking place in these coculture experimental settings, at least partly reproducing interactions occurring in vivo between the placenta and the endometrium.

Analysis of the EVCT invasion of the endometrial stromal cells

To track the two cell types interacting in the coculture system, the trophoblastic villi and endometrial fibroblasts were labeled, respectively, with red and green CellTracker dyes before setting up the coculture. Attachment of the trophoblastic villi to the endometrial fibroblast layer was let to occur for 16 h at which time phase-contrast pictures of the coculture were taken. Another set of phase-contrast pictures was taken 24 h later (Fig. 4, A-C). Cultures were fixed, labeled with Hoechst, and analyzed by confocal laser-scanning microscopy. Trophoblastic villi as well as the EVCTs produced by the stained trophoblast explants showed incorporation of the red CellTracker. This allowed their clear distinction from the green CellTracker-stained endometrial fibroblasts and enabled the visualization of the trophoblastic interstitial invasion of endometrial fibroblasts (Fig. 4, D-H). The reverse CellTracker incorporation combination (i.e. green CellTracker-stained villi and red CellTracker-stained endometrial fibroblasts) was also tested showing similar EVCT invasion patterns (data not shown). To confirm the specificity of the CellTrackers, we labeled the trophoblastic villi with the green CellTracker and immunostained the cocultures for either cytokeratin 7, HLA-G, or vimentin. As expected, the cytokeratin 7 and HLA-G labeling was found to fully match with the vital dye stainings (Fig. S1, A andB, published as supplemental data on The Endocrine Society's Journals Online web site at http:// endo.endojournals.org), whereas, on the other hand, vimentin and CellTracker stainings were exclusive of one another (Fig. S1,C). These observations showed the specificity of the Cell-Tracker dye and its suitability for identifying trophoblastic cells amid fibroblasts. The reverse experiment, involving prestained endometrial fibroblasts, was also performed and likewise showed optimal correlation with fibroblast markers (data not shown). Altogether these observations fully validated the use of CellTrackers for identifying, respectively, extravillous cytotrophoblasts and endometrial fibroblasts.

Regulation of trophoblastic migration/invasion by TGF␤: role of the RhoA-ROCK pathway

A role for TGF␤ in controlling trophoblastic invasion has been documented in various experimental settings. We first investigated whether our in vitro coculture system was amenable to reproduce this effect. The trophoblastic explants and endometrial fibroblasts were respectively labeled with the red and green CellTrackers and trophoblastic invasion was let to take place for about 16 h. At that time, TGF␤1 was added for 24 h, and effects of this cytokine were analyzed by both phase-contrast and confocal immunofluorescence microscopy (Fig. 4, bottom panel). Two major TGF␤1-related effects were observed: 1) blockade of EVCT migration within the fibroblast layer, resulting in the formation of stacks of EVCTs and 2) formation of large lacunes devoid of both EVCTs and endometrial fibroblasts. Similar effects were observed for the TGF␤2 and TGF␤3 isoforms (data not shown). Because both EVCTs and endometrial fibroblasts constitutively express TGF␤ (see above and our own unpublished RT-PCR data), we tested the effect on EVCT migration of anti-TGF␤ antibodies that prevent binding of all three TGF␤ isoforms to the receptor.

We also tested the effect of an inhibition of the Rho-ROCK signaling pathway, using the Y27632 inhibitor. Quite interestingly, both treatments induced an enhanced superficial trophoblastic migration that appeared to occur at the expense of interstitial invasion of the fibroblast layer. Indeed, extravillous cytotrophoblast migration exhibited a lesser dependence on the fibroblast orientation patterns as EVCTs were also able to migrate across transversally oriented endometrial fibroblasts (Fig. 5A, bottom panels). Y27632-dependent inhibition of ROCK was linked to an elongated EVCT cell morphology, as shown by cytokeratin 7 staining (Fig. 5B). In addition, confocal analysis of cocultures of prestained EVCTs and endometrial fibroblasts showed that in presence of Y27632, migration of EVCTs had a lesser impact on the endometrial fibroblast layer integrity, compared with control conditions or TGF␤1-treated cultures (Fig. 5C). Whereas TGF␤1 treatment led to the disappearance of endometrial fibroblasts after passage of EVCTs, in Y27632 conditions, the cell morphology of the endometrial fibroblasts apparently remained unchanged. Because of the variability between villi, inherent to the use of human specimens, we extended our survey of the effects of TGF␤1 and Y27632 treatments to a total of five different placentas, analyzing about 40 villi for each of the three conditions (TGF␤1, Y27632, or control) (Fig. 6). For each placenta considered, we evaluated the effect of the above-mentioned treatments on two main characteristics of the cocultures: 1) EVCT migration capacity (Fig. 6, A-C) and 2) size of lacunes made by EVCTs within the endometrial fibroblast layer (Fig. 6D). As previously observed (see Figs. 1, 2, and4), EVCT migration in the control conditions took place preferentially along the fibroblast longer orientation axis (migration dependent on the fibroblast orientation pattern). Treatment with TGF␤ appeared to frequently block EVCT migration, whereas, on the contrary, Y27632 treatment seemed to induce EVCT migration, whatever the fibroblast orientation pattern (migration independent of the fibroblast orientation pattern).

The statistical significance of these data were determined first by two-way ANOVA tests. They showed that for each type of migration, dependent on the fibroblast orientation pattern (Fig. 6A), blocked (Fig. 6B) or independent of the fibroblast pattern (Fig. 6C), the effects of the treatments were significant (P ϭ 0.032, 0.0001, and 0.003, respectively). On the other hand, the effects of the placentas (variations in the distributions due to the placental origin) were not considered significant (P values ranging from 0.52 to 0.98). The villi distributions in the various culture conditions were then compared using unpaired Student's t tests, which confirmed our initial observations. Indeed, in control conditions 70% of the trophoblastic villi were found to produce EVCTs that migrated along the fibroblast longer axis (Fig. 6A). Both TGF␤1 and Y27632 treatments gave rise to significantly different EVCT migration preferences (P ϭ 0.008 and 0.015, respectively) because this dependent migration was followed by only 20% of the villi. On the other hand, blockade of EVCT migration was observed in roughly 80% of the villi of the TGF␤1-treated cultures, a value significantly different from that of the control (P ϭ 0.001 in Fig. 6B). Finally, EVCT migration independently of the fibroblast orientation pattern was a feature that was observed mostly in Y27632-treated cultures (75% of the villi) and only 15% of the control cultures (P ϭ 0.004 in Fig. 6C). TGF␤1 rarely induced this type of EVCT migration (P ϭ 0.078). The size of lacunes formed within the fibroblast layer after EVCT migration was also found to change in response to the coculture treatment. La-FIG. 4. Interstitial trophoblastic invasion of endometrial fibroblasts. Inhibitory effect of TGF␤ on trophoblastic cell migration. Placental explants and endometrial fibroblasts were labeled beforehand with the red and green CellTrackers, respectively. They were subsequently cocultured for about 16 h. Phase-contrast pictures were taken (T0). Cultures were further incubated for 24 h at which time phase-contrast pictures were taken again (T24 h). Cultures were fixed and nuclei were stained with Hoechst. Phase-contrast micrographs are shown at times T0 and T24 h (A and B). C, High magnification of B; scale bar, 100 m. Confocal microscopy sections of the fields shown in C are shown in low magnification (D, scale bar, 100 m) and high magnification (E-H, scale bar, 50 m). Top panels, Control conditions. Bottom panels, TGF␤ treatment. At time T0, cultures were treated with TGF␤1 (10 ng/ml) and further incubated for 24 h at which time phase-contrast pictures were again taken (TGF␤ T24 h). Cultures were fixed and analyzed as for control conditions.

cunes were found to slightly increase with TGF␤1 treatment, even though the difference with the control was not considered significant by Mann-Whitney evaluation (Fig. 6D, P ϭ 0.322). On the other hand, Y27632 treatment considerably limited the size of lacunes compared with either control or TGF␤1 conditions (P ϭ 0.003 and 0.002, respectively), an observation consistent with the more superficial migration of EVCTs in these culture conditions.

Discussion

Cocultures of human trophoblastic villi and primary endometrial fibroblasts were used in this work to characterize trophoblast migration. Extravillous cytotrophoblast migration was followed by time-lapse phase-contrast microscopy. Trophoblast explants, as opposed to isolated cytotrophoblast cells or immortalized EVCT cell lines, were used, and therefore, we analyzed the migration of EVCTs produced con-temporaneously during the coculture. The data shown are based on experiments performed on a total of 20 different placentas obtained at very early pregnancy (4 -7 wk). The young age of these placentas enabled us to observe migration phenomena such as formation of cytotrophoblast cell columns within very short time frames (16 -48 h after seeding the explants), compared with other reports in which the use of older placentas (8 -12 wk) led to delayed trophoblast migration (of the order of 76 h) [START_REF] Vicovac | Trophoblast differentiation during formation of anchoring villi in a model of the early human placenta in vitro[END_REF][START_REF] Aplin | Development of cytotrophoblast columns from explanted first-trimester human placental villi: role of fibronectin and integrin ␣5␤1[END_REF]. Boyden chambers are often used for three-dimensional migration assays that measure motility as the number of cells that migrate across a filter separating two culture compartments. They have provided valuable information on the migration of a number of strains, including transformed cell lines. However, this technique mostly relies on artificial matrices and accordingly does not take into account interactions with the normal cellular microenvironment. This limitation led us to set up a system FIG. 5. Effect of exogenous TGF␤, ROCK inhibitor Y27632, and anti-TGF␤ antibodies on extravillous trophoblast cell invasion. Placental explants and endometrial fibroblasts were cocultured for about 16 h. Phase-contrast pictures were taken of fields showing a beginning of trophoblastic invasion (T0). Cultures were treated with TGF␤1 (10 ng/ml), the ROCK inhibitor Y27632 (5 M), or anti-TGF␤ antibodies (4 g/ml) or left untreated (Ctl) and further incubated for 24 h, at which point phase-contrast pictures of the same fields were taken (T24 h). A, Phase-contrast micrographs of a representative experiment performed with trophoblastic villi obtained from the same placenta. B, At time T24 h, cultures were fixed and stained for cytokeratin 7 (CK7) and Hoechst (shown in the overlays). C, Before the coculture, the placental explants and endometrial fibroblasts were labeled with CellTrackers. They were otherwise treated as in A. EVCTs and endometrial fibroblasts are identified as red and green cells, respectively. The Hoechst staining is shown in the overlays. whereby stromal endometrial fibroblasts are used as a matrix for trophoblastic cell migration. The observed trophoblastinduced decidualization of the endometrial fibroblasts indicated that our coculture system could recapitulate interactions that occur in vivo between the placenta and the endometrial stroma, giving further support to the use of human primary endometrial fibroblasts for studying trophoblast invasion.

Extravillous cytotrophoblast migration was followed by phase-contrast time-lapse microscopy and fluorescence microscopy of vitally stained cells. EVCTs were found to migrate in nearly straight paths away from the villi. However, individual EVCT migration trajectories were clearly influenced by the underlaying endometrial fibroblast orientation pattern and showed a strong preference for a migration along the fibroblast longer axis. Interestingly, EVCT migration characteristics differed, depending on the tissue origin of the fibroblasts. When derm instead of endometrium primary fibroblasts (isolated from the same donor) were used in the coculture, EVCT migration was found to occur more superficially on the fibroblast layer (our unpublished data). These observations further emphasized the role of the fibroblast matrix for EVCT migration and could be related to recent data showing that different repertoires of genes are expressed in fibroblasts, depending on the tissue they originate from [START_REF] Rinn | Anatomic demarcation by positional variation in fibroblast gene expression programs[END_REF]. EVCT velocities in our coculture system ranged from 10 to 40 m/h with an average measured velocity of 23 m/h. These values are close to velocities found for carcinomas in three-dimensional collagen matrices, ranging from 8 to 14 m/h [START_REF] Niggemann | Tumor cell locomotion: differential dynamics of spontaneous and induced migration in a 3D collagen matrix[END_REF][START_REF] Heyder | Role of the ␤1-integrin subunit in the adhesion, extravasation and migration of T24 human bladder carcinoma cells[END_REF], or for keratinocytes (42 m/h) [START_REF] Frank | Laminin 5 deposition regulates keratinocyte polarization and persistent migration[END_REF]. They are much lower, however, than velocities found for lymphocytes, of the order of 400 m/h [START_REF] Boissonnas | In vivo imaging of cytotoxic T cell infiltration and elimination of a solid tumor[END_REF][START_REF] Garrod | Regulation of Trophoblast Migration by TGF␤ patrol peripheral lymph nodes forming stable conjugates to eliminate MHCmismatched targets[END_REF].

We analyzed the effect of TGF␤ on EVCT migration by time-lapse phase-contrast microscopy and confocal microscopy of fluorescently labeled trophoblasts and endometrial fibroblasts. TGF␤ was previously reported to block EVCT migration [START_REF] Irving | Functional role of cell surface integrins on human trophoblast cell migration: regulation by TGF-␤, IGF-II, and IGFBP-1[END_REF][START_REF] Graham | Mechanism of control of trophoblast invasion in situ[END_REF][START_REF] Godkin | Transforming growth factor ␤ and the endometrium[END_REF][START_REF] Rama | TGF␤1 induces multiple independent signals to regulate human trophoblastic differentiation: mechanistic insights[END_REF][START_REF] Karmakar | Modulation of ezrin and E-cadherin expression by IL-1␤ and TGF-␤1 in human trophoblasts[END_REF](17)[START_REF] Jones | TGF-␤ superfamily expression and actions in the endometrium and placenta[END_REF]. We found a similar effect of TGF␤ in our coculture system, and all three TGF␤ isoforms, TGF-␤1, TGF-␤2, and TGF-␤3, were found to inhibit EVCT invasion of the endometrial fibroblasts. Besides, TGF␤ treatment induced the formation of compact EVCT stacks up to 20 m in height, as observed by confocal microscopy. Formation of these stacks was correlated with increased E-cadherin expression at the EVCT plasma membrane (data not shown). TGF␤ treatment also induced EVCTs to form large lacunes, devoid of cells, within the endometrial fibroblast layer by a mechanism that still needs to be determined. Because murine trophoblast cells were reported to phagocyte cells from the endometrium (44), we looked for such a phenomenon using vitally stained cells in our coculture system. However, we were unable to provide evidence that such an engulfment mechanism was taking place between human extravillous trophoblastic cells and endometrial fibroblasts. Alternatively, metalloproteinases secreted by the trophoblast might also be involved [START_REF] Staun-Ram | Human trophoblast function during the implantation process[END_REF][START_REF] Cohen | Metalloproteinases and human placental invasiveness[END_REF]. Determining their putative role in the formation of the lacunes observed will need further investigation.

Previous reports showed a role for the RhoA/ROCK pathway in supporting trophoblast migration for both primary EVCTs and immortalized trophoblastic cell lines (28, [START_REF] Shields | Rho GTPases differentially regulate IGF1 receptor-dependent and -independent actions of IGFII on human trophoblast migration[END_REF][START_REF] Nicola | Roles of Rho guanosine 5Ј-triphosphataseA, Rho-kinases and extracellular signal regulated kinase (ERK1/2) in prostaglandin E2-mediated migration of first trimester human extravillous trophoblast[END_REF]. This was tested in Boyden chamber-type assays in which migration efficiency is scored as the number of cells crossing a synthetic matrix. These assays are obviously very different, in their biochemical and biophysical properties, from the system we used that involved an endometrial fibroblastbased matrix. Nevertheless, we observed, as they did, that addition of the chemical ROCK inhibitor Y27632 in the coculture disrupted the actin stress fibers found in untreated EVCTs, leading to cortical relocalization of F-actin (data not shown), and induced an elongated EVCT morphology. However, in contrast to their observations, we found that EVCT migration was not abolished in Y27632-treated cocultures. Unexpectedly, inhibition of the RhoA-ROCK pathway promoted efficient EVCT two-dimensional migration. This increased migration capacity was apparently linked to weakened interactions between EVCTs and the endometrial fibroblasts because EVCT migration showed a lesser dependence on the fibroblast orientation pattern and because, conversely, the fibroblast layer integrity was maintained after passage of the EVCTs. ROCK activity has been shown to be important for the migration of tumor cells, in particular in the context of amoeboid cell motility [START_REF] Itoh | An essential part for Rho-associated kinase in the transcellular invasion of tumor cells[END_REF][START_REF] Sahai | Illuminating the metastatic process[END_REF]. However, different modes of cell migration with distinct requirements for Rho/ ROCK signaling could be found in tumor cells [START_REF] Sahai | Differing modes of tumour cell invasion have distinct requirements for Rho/ROCK signalling and extracellular proteolysis[END_REF]. Whereas ROCK activation was required for bleb-associated motility, elongated protrusive movements were found to occur independently of ROCK. Recent reports also established that ROCK activation could be required in the stromal fibroblasts supporting invasion but not in the actual invasive tumor cells, as shown for squamous cell carcinoma cells [START_REF] Gaggioli | Fibroblast-led collective invasion of carcinoma cells with differing roles for RhoGTPases in leading and following cells[END_REF]. Whether such phenomena occur in our coculture system and could explain why treatment with the Y27632 inhibitor induces a switch between threedimensional and two-dimensional trophoblast migration will require further investigation.

FIG. 1 .

 1 FIG. 1. Invasion of endometrial fibroblasts by trophoblastic cells produced by placental explants. A, Time-lapse analysis of the migration of cytotrophoblast cells on confluent layers of endometrial fibroblasts. Frames corresponding to time-lapse phase-contrast images show EVCT migration at the indicated times. Scale bar, 100 m. B, Trajectories and kinetics of three independent EVCTs within the endometrial fibroblast layer. Kinetics curves shown in panels I, II, and III refer to the corresponding trajectories in the phasecontrast micrograph.

FIG. 2 .

 2 FIG. 2. Invasion of EVCTs on endometrial stromal cells. Kinetics of migration are shown. A, The production and migration of extravillous trophoblastic cells was followed by time-lapse phase-contrast microscopy with camera-scanned fields. The phase-contrast micrographs show image reconstitution from the 5 ϫ 5 contiguous fields (total of 25 fields) from the time-lapse images taken at the indicated times. Scale bar, 1 mm. B, The migrating kinetics (shown in panels I, II, and III) were determined for the three corresponding EVCT migrating fronts whose trajectories are indicated in the phase-contrast micrograph. Scale bar, 1 mm.

FIG. 3 .

 3 FIG. 3. Decidualization of endometrial fibroblasts. A, In vitro-induced decidualization of endometrial primary fibroblasts. Endometrial fibroblasts were treated with medroxyprogesterone acetate (1 M) and 8-bromoadenosine-cAMP (0.5 mM) for 4 d. Cells were fixed and stained for PRL and IGFBP1. Fibroblasts were counterstained with vimentin and Hoechst (triple staining in the overlay panels). Scale bar, 50 m. Ctl, Control; Dec, decidualization conditions. B, Trophoblast-induced decidualization of endometrial fibroblasts. Endometrial fibroblasts were cocultured with placental explants for 48 h. Cell cultures were then fixed and stained for PRL, vimentin, and Hoechst (triple staining in the overlay panels). The mock labeling was done in the absence of the PRL antibody. Scale bar, 50 m.

  FIG.5. Effect of exogenous TGF␤, ROCK inhibitor Y27632, and anti-TGF␤ antibodies on extravillous trophoblast cell invasion. Placental explants and endometrial fibroblasts were cocultured for about 16 h. Phase-contrast pictures were taken of fields showing a beginning of trophoblastic invasion (T0). Cultures were treated with TGF␤1 (10 ng/ml), the ROCK inhibitor Y27632 (5 M), or anti-TGF␤ antibodies (4 g/ml) or left untreated (Ctl) and further incubated for 24 h, at which point phase-contrast pictures of the same fields were taken (T24 h). A, Phase-contrast micrographs of a representative experiment performed with trophoblastic villi obtained from the same placenta. B, At time T24 h, cultures were fixed and stained for cytokeratin 7 (CK7) and Hoechst (shown in the overlays). C, Before the coculture, the placental explants and endometrial fibroblasts were labeled with CellTrackers. They were otherwise treated as in A. EVCTs and endometrial fibroblasts are identified as red and green cells, respectively. The Hoechst staining is shown in the overlays. B and C, Confocal microscopy sections of the cultures are shown (scale bar, 50 m).

FIG. 6 .

 6 FIG. 6. Statistical analysis of the effects of TGF␤ and Y27632 on trophoblast migration. Two main trophoblast invasion criteria were analyzed: migration (A-C) and formation of lacunes (D). A, Distribution of trophoblastic villi producing EVCTs that migrate preferentially along the longer fibroblast axis (migration dependent on the fibroblast orientation pattern). B, Distribution of villi showing a blockade in EVCT migration upon culture treatment. C, Distribution of villi producing EVCTs whose migration is independent on the fibroblast orientation pattern. In the box and whisker diagrams, whiskers mark 10th and 90th percentile, boxes mark 25th and 75th percentile. P values were determined with unpaired Student's t tests. D, Quantification of lacune length in the different culture conditions, shown are the median values of the distributions Ϯ SD. Data were obtained from experiments performed on five different placentas and at different times. These placentas provided material to analyze a total of 16 independent culture plates with a total of 48 trophoblastic villi for the control conditions, 12 plates, and 36 villi for the TGF␤ conditions and 10 plates and 37 villi for the Y27632-treated cultures.

  Fafet et al. • Regulation of Trophoblast Migration by TGF␤ Endocrinology, September 2008, 149(9):4475-4485 4477

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Acknowledgments

We thank the Montpellier RIO imaging facility (MRI) for providing adequate environment for time-lapse analysis and confocal microscopy. The authors also thank the staff of the Obstetrics Department of Hospital Arnaud de Villeneuve (Montpellier) for providing placentas and endometrial specimens. We thank Thierry Gostan for performing statistical analysis and Jean Marie Blanchard, Delphine Haouzi, Robert Hipskind, Marion Peter, and Pierre Savagner for helpful discussion and critical reading of the manuscript.

Received February 22, 2008. Accepted May 13, 2008. Address all correspondence and requests for reprints to: Marie-Luce Vignais, Institut de Ge ´ne ´tique Mole ´culaire de Montpellier, 1919 Route de Mende, 34293 Montpellier cedex 5, France. E-mail: vignais@igmm. cnrs.fr.

This work was supported by Grant 3446 from the l'Association pour la Recherche sur le Cancer (to M.-L.V.).

Disclosure Statement: The authors have nothing to disclose.


C Alabert

L Rogers

L Kahn

S Niellez

P Fafet

S Cerulis

RA J M Blanchard

Dr M-L Vignais
email: vignais@igmm.cnrs.fr

Cell type-dependent control of NF-Y activity by TGF-b

Keywords: TGF-b, NF-Y, MAPK

Transforming growth factor b (TGF-b) is a pluripotent cytokine that regulates cell growth and differentiation in a cell type-dependent fashion. TGF-b exerts its effects through the activation of several signaling pathways. One involves membrane proximal events that lead to nuclear translocation of members of the Smad family of transcriptional regulators. TGF-b can also activate MAPK cascades. Here, we show that TGF-b induces nuclear translocation of the NF-YA subunit of the transcription factor NF-Y by a process that requires activation of the ERK cascade. This results in increased binding of endogenous NF-Y to chromatin and TGF-bdependent transcriptional regulation of the NF-Y target gene cyclin A2. Interestingly, the kinetics of NF-YA relocalization differs between epithelial cells and fibroblasts. NIH3T3 fibroblasts show an elevated basal level of phosphorylated p38 and delayed nuclear accumulation of NF-YA after TGF-b treatment. In contrast, MDCK cells show low basal p38 activation, higher basal ERK phosphorylation and more rapid localization of NF-YA after induction. Thus, NF-Y activation by TGF-b1 involves ERK1/2 and potentially an interplay between MAPK pathways, thereby opening the possibility for finely tuned transcriptional regulation.

Introduction

Transforming growth factor b (TGF-b) signaling is implicated in biological processes ranging from growth control to cell differentiation and is disrupted in many cancers (Siegel and Massague, 2003;Waite and Eng, 2003;Yingling et al., 2004). The biological outcome of TGF-b exposure depends on both the cellular context and environmental cues. The effects of TGF-b are triggered by binding of the ligand to its cell surface receptor, which leads to the formation of the active receptor kinase and to phosphorylation of downstream targets (de Caestecker, 2004). Several signaling pathways have been identified downstream of TGF-b receptors, among which that involving the Smad family of transcription factors is the best characterized. TGF-b can also activate the ERK, p38 and SAPK/JNK mitogen activated protein kinase (MAPK) cascades to different extents, depending on cell type (Attisano and Wrana, 2000;Derynck and Zhang, 2003). One potential intermediate is TGF-b-activated kinase 1 (TAK1) (Yamaguchi et al., 1995), a MAPKKK that can activate the JNK (Wang et al., 1997) or p38 pathway (Moriguchi et al., 1996).

Outside of Smads, few transcription factors are known to be targeted by TGF-b. We focused our study on the ubiquitous and evolutionarily conserved transcription factor NF-Y, also known as CCAAT-binding factor (CBF). NF-Y is composed of three subunits: NF-YA, NF-YB and NF-YC (Maity et al., 1992). NF-YB and NF-YC contain histone-fold motives that are required for dimerization, as well as association with NF-YA, and all three subunits are necessary for specific-DNA binding (Sinha et al., 1995). While the large glutamine-rich domains of the NF-YA and NF-YC subunits can activate transcription (Coustry et al., 1996), NF-Y can also mediate repression of target genes (Manni et al., 2001). NF-Y helps control the expression of many genes, including the cell cycle regulatory genes cyclin A2, cyclin B2 and E2F1 (Hu and Maity, 2000;Manni et al., 2001). Although NF-Y was originally considered a constitutive transcription factor, recent data indicate that its activity is regulated, in particular through NF-YA (Mantovani, 1999;Marziali et al., 1999). The knockout of NF-YA leads to early embryonic lethality and accordingly NF-YA À/À mouse embryonic fibroblasts show a block in cell growth, confirming a role for NF-Y in regulating cell proliferation (Bhattacharya et al., 2003).

In this study, we show that NF-Y is a target of TGF-b signaling. TGF-b treatment leads to localization of NF-YA to the nucleus. This nuclear accumulation correlates with increased binding of endogenous NF-Y to chromatin and transcriptional regulation as shown for the cyclin A2 gene. NF-YA localization is dependent upon ERK activation in both fibroblasts and epithelial cells. However, the kinetics of NF-YA nuclear accumulation differs in these two cell types, a difference that may be linked to the basal level of activation of both the ERK and p38 cascades. An interplay between MAPK cascades would allow a cell type-dependent fine tuning of the expression of key cell cycle genes, such as cyclin A2, thereby providing specificity for the TGF-b response.

Results

NF-YA localizes to the nucleus in response to TGF-b Results from our laboratory have previously implicated the trimeric transcription factor NF-Y in the transcriptional regulation of the cyclin A gene (Plet et al., 1997) and preliminary data suggested its likely involvement in the transcriptional response to TGF-b (data not shown). Therefore, we investigated whether this involved a change in the subcellular localization of NF-Y similar to the TGF-b-dependent nuclear translocation of the Smad proteins (Derynck and Zhang, 2003;Shi and Massague, 2003;ten Dijke and Hill, 2004). Our study focused on the NF-YA subunit, since it provides the DNA binding specificity to the trimeric transcription factor. Moreover, the A subunit is regulated in numerous situations (Mantovani, 1999). NIH3T3 fibroblasts were treated with TGF-b and then processed for indirect immunofluorescence at various times in order to visualize changes in the cellular localization of endogenous NF-YA and Smad3 (Figure 1). As expected, Smad3 accumulated in the nucleus within 15 min of TGF-b treatment (Figure 1a). NF-YA localized within the cytoplasm and nucleus in cycling cells and then became nuclear in response to TGF-b. Interestingly, NF-YA nuclear localization peaked at around 90 min of TGF-b treatment with kinetics that clearly differed from Smads, for which the peak occurred at 15 min. In contrast, NF-YB localization was restrained to the nucleus in NIH3T3 cycling cells and remained so during TGF-b treatment (Supplementary data). To confirm the nuclear relocalization of NF-YA and Smad3 biochemically, western blots of nuclear extracts from TGF-btreated NIH3T3 cells were incubated with antibodies against these proteins (Figure 1b). Consistent with the results above, NF-YA levels progressively increased in the nucleus after TGF-b treatment but with delayed kinetics relative to Smad3. Quantification of the nuclear accumulation of Smad3 and NF-YA was performed from blots obtained from three independent sets of protein extracts. It showed increases in nuclear concentrations of respectively 5.1-fold for Smad3 (s.d.: 0.7) and 2.3-fold for NF-YA (s.d.: 0.2). This nuclear accumulation reflected translocation of the cytoplasmic pool of NF-YA, since TGF-b induction did not alter the total amount of the protein (Figure 1b).

TGF-b-induced NF-YA nuclear accumulation correlates with increased binding to chromatin of CCAAT boxcontaining promoters and with transcriptional regulation

To demonstrate a functional role for the nuclear relocalization of NF-YA, we first investigated whether this correlated with increased binding to its target promoters. As the transcription factor NF-Y can modify chromatin structure and associate with both histones and other transcription factors, we tested this using chromatin immunoprecipitation (ChIP). Chromatin was prepared from TGF-b-treated NIH3T3 fibroblasts and immunoprecipitated with antibodies specific for NF-YA. To follow NF-YA binding, we used both semiquantitative and real-time PCR to detect the inverted CCAAT box of the cyclin A2 promoter that binds NF-Y in vitro and in vivo (Plet et al., 1997) (Figure 2a andb). TGF-b stimulation led to increased recruitment of NF-YA to the CCAAT box but not to an upstream region of the cyclin A promoter (see table ). Likewise, only background levels of cyclin A CCAAT box chromatin were precipitated by an irrelevant antibody against the tyrosine kinase Tyk2. These data indicate that TGF-b induces increased NF-YA binding to the cyclin A CCAAT box in vivo. In addition, TGF-b treatment also resulted in increased DNA binding of the NF-YB subunit, an effect that was abolished when, instead of the wild-type (wt) NF-YA, a dominant negative mutant of NF-YA was overexpressed (data not shown). Similarly NF-YA showed increased binding, in response to TGF-b, to the CCAAT boxes present in the promoters of the cyclin B2 and E2F1 genes (Bolognese et al., 1999) (Figure 2c). There was only background binding to the promoter of the housekeeping gene hypoxanthine phosphoribosyltransferase (HPRT) that contains no CCAAT box. These data confirm that TGF-b signaling results in increased DNA recruitment of NF-YA on CCAAT boxes in vivo. Besides, we also tested NF-Y binding by gel shift assay using nuclear extracts from mock or TGFb-treated cells. It also showed increased NF-Y DNA binding activity in response to TGFb (data not shown).

We used the cyclin A2 promoter to study the activity of NF-Y in response to TGF-b since this gene is repressed by TGF-b in CCL39 cells (Barlat et al., 1993(Barlat et al., , 1995)). Moreover, NF-Y binds to the inverted CCAAT box found near the cyclin A2 transcription initiation sites (Plet et al., 1997) and here, we have shown that TGF-b induces NF-Y binding to this site in chromatin. We compared the activities of cyclin A2 promoters bearing either a wt or a mutated (mut) CCAAT box, using luciferase reporter genes in transient transfection assays. Notably, our cyclin A2 promoter constructs are mutated at the CCRE-CHR site to bypass cell cycledependent regulation of the cyclin A2 gene conferred by this site (Huet et al., 1996;Philips et al., 1999;Fajas et al., 2001). We observed a twofold transcriptional repression by TGF-b of the wt cyclin A2 CCAAT box that dropped to 1.3 upon mutation of the CCAAT box (Figure 2d). These differences, obtained from four independent series of experiments, were significant (P-valueo0.0001). Likewise, overexpression of the wt and of a dominant negative mutant of NF-YA had opposing effects on reporter activity (Po0.0005), as the NF-YA dominant negative mutant diminished while the wt protein increased repression of the cyclin A2 by TGF-b (Po0.05). These data indicate that the CCAAT box and NF-Y participate in transcriptional regulation of the cyclin A2 gene in response to TGF-b, therefore supporting a functional role for nuclear localization of the NF-YA subunit in response to TGF-b.

TGF-b-induced nuclear accumulation of NF-YA does not require protein neosynthesis or the Smad2/Smad3 signaling pathway NF-YA levels did not change in whole cell extracts in response to TGF-b (Figure 1b). However, its delayed accumulation in the nucleus relative to Smads could be dependent on prior activation of the Smads and/or de novo synthesis of other signaling components. To test the latter, we pretreated NIH3T3 cells with cycloheximide prior to incubation with TGF-b. Inhibition of protein synthesis did not hinder TGF-b-driven nuclear accumulation of NF-YA (Figure 3a). To test the role of Smad activation, we transfected an expression vector encoding Smad7, which inhibits Smad2/3 signaling (data not shown and (Derynck and Zhang, 2003)). It slightly enhanced TGFb-dependent phosphorylation of ERK (Supplementary Figure 2). TGF-b still induced nuclear accumulation of NF-YA in Smad7 overexpressing cells while, as previously documented, the recombinant Smad7 protein redistributed to the cytoplasm (Figure 3b). These data indicate that NF-YA nuclear accumulation in response to TGF-b is independent of Smad activation and protein neosynthesis.

NF-YA nuclear accumulation depends on ERK activation

In numerous cultured cell lines, MAPK cascades mediate some TGF-b intracellular signaling events. Therefore, we tested for MAPK activation by TGF-b in our NIH3T3 fibroblasts and whether MAPKs played a role in NF-YA nuclear localization. TGF-b stimulated activating phosphorylation of ERK2 by 15 min and again by 60 min after treatment but did not alter the level of ERKs 1 and 2 (Figure 4a). Activating phosphorylation of p38 in response to TGF-b treatment clearly differed from that of ERK, with a progressive decrease up to 30 min after TGF-b treatment and a sharp transient peak at the 60 min time point. As with ERK1/2, p38 levels remained unaffected by TGF-b addition (Figure 4a). Notably, TGF-b did not activate SAPKs in NIH3T3 cells, while they, as well as ERK1/2 and p38, were readily induced by subinhibitory levels of anisomycin (Figure 4a).

To evaluate the role of these MAPKs in NF-YA nuclear accumulation, we used pharmacological inhibitors specific for the ERK and p38 cascades. PD98059 blocks activation of MEK1 and thereby ERK1/2 (Alessi et al., 1995), while SB203580 inhibits p38a and b activity but not their activating phosphorylation (Young et al., 1997). As expected, PD98059 diminished TGF-b-driven phosphorylation of ERK; SB203580 inhibited phosphorylation of ATF1, a well-characterized target of the p38 cascade (Tan et al., 1996) (Figure 4b) and thus an indicator of p38 kinase activity. SB203580 slightly increased SAPK phosphorylation after TGF-b treatment, suggesting that, like in other cell systems, p38 inhibition enhances TAK1 induction and thereby that of SAPK (Cheung et al., 2003).

We next examined the effects of these inhibitors on TGF-b-dependent accumulation of NF-YA in the nucleus. This was blocked by pretreatment of cells with PD98059 but not by the SAPK inhibitor SP600125 (Figure 4c). These data were quantified from two independent experiments, using either FITC-or cy3conjugated antibodies. While the proportion of cells harboring a high nuclear concentration of NF-YA significantly increased from 16 to 78% (s.d. 4.2) upon TGFb treatment, pretreatment of the NIH3T3 cells with PD98059 prevented this increase and NF-YA remained spread between the nucleus and the cytoplasm in 90% (s.d. 5.7) of the cells. Similar results were obtained in the CCL39 fibroblasts (data not shown). In the presence of SB, NF-YA was found around the nuclear periphery after TGF-b induction. This could reflect a failure of NF-YA to localize to the nucleus after TGF-b treatment or an accelerated nuclear export. Consistent with the second possibility are preliminary observations that NF-YA appears more rapidly in the nucleus upon TGF-b induction in cells pretreated with SB alone or together with SP, which blocks SB-induced SAPK activation. We also tested the effect of okadaic acid, a specific inhibitor of type 1 and 2A serine/ threonine phosphatases, as PP2A was both proposed to be involved in TGF-b signaling and to regulate the activities of ERK and p38 (Janssens and Goris, 2001). Okadaic acid activated all three MAPK pathways and led to NF-YA nuclear accumulation independently of TGF-b stimulation, thus emphasizing the important role of serine/threonine phosphorylation in NF-YA nuclear localization (Figure 4b andc). Interestingly, Smads remained cytoplasmic upon okadaic acid treatment (Figure 4c), providing further evidence that NF-YA nuclear accumulation in response to TGF-b does not require prior Smad3 activation. Taken together, these data show that (1) TGF-b induces NF-YA nuclear accumulation in NIH3T3 fibroblasts with kinetics delayed compared to those of the Smads, and (2) this is dependent upon activation of the ERK1/2 cascade but not that of Smads.

The kinetics of TGF-b-dependent NF-YA nuclear accumulation differ between epithelial cells and fibroblasts

As TGF-b has distinct effects depending on cell type, we investigated whether it could also induce nuclear accumulation of NF-YA in epithelial cells. Interestingly, TGF-b also induced nuclear localization of NF-YA in MDCK epithelial cells that was faster and more transient than that observed in NIH3T3 fibroblasts. The diffuse NF-YA cellular signal observed in cycling MDCK cells concentrated in the nucleus within 15 min of TGF-b treatment and then redistributed within the cell (Figure 5a). Although the basal level of ERK phosphorylation was higher in these cells, it still showed a relative increase at 15 and 60 min after TGF-b addition (Figure 5b). Similar to NIH3T3 cells, inhibition of the ERK cascade with PD98059 blocked nuclear accumulation of NF-YA but not that of Smad3 (Figure 5c). Quantification of these data was done from two independent experiments, using either FITC-or cy3-conjugated antibodies, on a total of 200 cells for each condition. TGFb treatment led to an increase from 7 to 74% (s.d. 3.5) of the proportion of cells harboring a high NF-YA nuclear concentration that was reduced to 13% in the presence of PD98059. Importantly, we obtained the same data in the human epithelial cell line HEK treated with TGF-b (data not shown). Curiously, in PD-treated MDCK cells, NF-YA localized at the membrane, an effect that remains yet to be explained.

To confirm the inhibitory effect of PD98059 on the nuclear relocalization of NF-YA biochemically, nuclear extracts were prepared from TGF-b-treated MDCK cells in the presence or absence of PD98059, separated by SDS-PAGE and the Western blots were incubated with antibodies against this protein (Figure 6a). Consistent with the results above, NF-YA levels rapidly increased in the nucleus after TGF-b treatment with kinetics similar to those of Smad3. This increase was blocked by pretreatment with PD98059, an inhibitory effect that was not observed for Smad3. Quantification of the nuclear accumulation of NF-YA (performed from two independent western blots) showed a 2.5-fold (s.d. 0.6) increase in nuclear NF-YA that returned to 1.0 (s.d. 0.1) in the presence of PD98059. This nuclear accumulation was followed by an increase in NF-YA association with chromatin that peaked at 30 min (Figure 6b). Chromatin solubilization by brief micrococcal nuclease (MNase) treatment showed that NF-YA was indeed associated with chromatin, as opposed to some other insoluble structure (Figure 6c). Pretreatment of the MDCK cells with PD98059 prevented this increase while Smad3 recruitment to chromatin in response to TGFb was unaffected by the pharmacological inhibitor. Association of NF-YA with chromatin was quantified from two independent chromatin preparations. It showed a 2.0-fold (s.d. 0.2) increase in nuclear NF-YA, while PD98059 blocked chromatin association (1.070.1). Together these data show that both TGFbinduced nuclear relocalization and chromatin recruitment of NF-YA depend on ERK activation.

Smads, like numerous other proteins, have been shown to constantly shuttle between the cytoplasm and the nucleus. To test whether this was the case for NF-YA, we treated both NIH3T3 and MDCK cells with leptomycin B (LMB), an inhibitor of CRM1-driven nuclear export. As expected, LMB treatment led to the sequestering of ERK in the nucleus (Adachi et al., 2000) (Figure 7a). In contrast, the endogenous NF-YA protein, which was found in both the nuclear and cytoplasmic compartments in the absence of LMB, appeared to accumulate in the cytoplasm in a number of NIH3T3 cells in the presence of LMB, an effect that was even more pronounced in MDCK cells (Figure 7a, top and bottom panels). These data were quantified from two independent experiments for each cell line, using either FITC-or cy3-conjugated antibodies to detect NF-YA (Figure 7b, left and right panels for respectively NIH3T3 and MDCK cells). To avoid any possible bias due to cell permeabilization conditions, NIH3T3 cells were also transfected with GFP-NFYA expressing vectors, in which case cell nuclei were Hoechst labeled directly following LMB treatment and fixation. Transfection led to a nuclear localization of the recombinant NF-YA fusion protein in a high proportion of the cells (84%), while it was evenly spread between the nucleus and the cytoplasm in the remaining 16% of cells. Even under these conditions, LMB treatment led to a cytoplasmic accumulation of NF-YA as the corresponding population increased from 16 to 36% (Figure 7b, middle panel). These data were obtained from three independent transfections, with a total cell count of 400 for each condition. Altogether, these data indicate that NF-YA is not retained in the nucleus by leptomycin treatment and accordingly that its nuclear export is independent of CRM1. In fact, LMB has the unexpected effect of increasing cytoplasmic accumulation of NF-YA. This observation confirms that the intracellular distribution of NF-YA is regulated and suggests furthermore that it depends on factors that are themselves controlled by CRM1.

Discussion

We show in this study that the NF-YA subunit of the transcription factor NF-Y, which is found in the nuclear and cytoplasmic subcellular compartments, accumulates in the nucleus upon TGF-b1 induction. Accordingly, it binds to chromatin regions containing CCAAT regulatory sequences in the promoters of several cell cycle regulatory genes. This TGF-b-dependent nuclear localization of NF-YA is observed in epithelial cells and fibroblasts, albeit with different kinetics, and is dependent upon activation of the ERK1/2 cascade. Notably, increased basal activation of the p38 cascade and low basal phospho-ERK1/2 levels seem to correlate with slower NF-YA nuclear accumulation in fibroblasts. Thus, the balance of activity of these two MAPK cascades might help control the dynamics of NF-YA nuclear localization, and thus its activity, induced by TGF-b1.

The nuclear accumulation of NF-YA in response to TGF-b is reminiscent of the nuclear shuttling of R-Smads. Translocation of phosphorylated Smad3 protein involves an interaction between its nuclear localizing signal (NLS) and importin-b1 (Xiao et al., 2000;Kurisaki et al., 2001). Although Smad2 contains a NLS region similar to that of Smad3 (Xiao et al., 2000), it appears to use a distinct translocation mechanism that is independent of the NLS and of importin-b, and instead involves direct interactions between Smad2 and the nucleoporins Nup214 and Nup153 (Xu et al., 2002). All three Smad proteins have been reported to constantly shuttle between the cytoplasm and the nucleus (Inman et al., 2002;Nicolas et al., 2004). The export of Smad4 was shown to be dependent of CRM1 while the subcellular distribution of Smad2 and Smad3 is unaffected by leptomycin B (LMB) treatment (Pierreux et al., 2000). After this manuscript was submitted, NF-YA was reported to contain a nonclassical nuclear localization signal (ncNLS) at its C-terminus and to be translocated to the nucleus by an importin b-mediated pathway (Kahle et al., 2005). It remains to be seen whether NF-YA also contains a nuclear export signal and whether it shuttles in and out of the nucleus like Smads. Thus, it is difficult at present to determine whether its nuclear accumulation actually arises from a change in shuttling dynamics. Curiously, leptomycin B treatment drove endogenous NF-YA to the cytoplasm, while it led to nuclear retention of ERK. A similar effect of LMB, leading to increased cytoplasmic localization, was observed for an ectopically expressed GFP-NFYA fusion protein. This provides further evidence that the subcellular localization of NF-YA is regulated and that the factors controlling this are themselves affected by inhibiting CRM-1-mediated nuclear export. In light of our data, it seems fair to speculate that ERK and p38 are among these regulatory factors. NF-YA was recently shown to be phosphorylated in vivo (Yun et al., 2003). This involved two serine-proline motifs that could be phosphorylated by cdk2 kinase. Notably, these two serine residues lie just C-terminal to the above-mentioned ncNLS (Kahle et al., 2005). Moreover, mutation of the two serine residues to alanine allowed formation of the NF-Y trimer but inhibited DNA binding. However, the intracellular localization of the NF-YA mutant was not addressed. Based on our data, we can speculate that these two serine residues might also be substrates for ERK and therefore their mutation would decrease the nuclear concentration of the NF-YA mutant. In addition, the observation that p38 can phosphorylate serine residues that are not directly followed by prolines (Bulavin et al., 2001;Cheung et al., 2003) opens the possibility for other p38 target sites on NF-YA. This remains to be investigated.

We show that, in contrast to the B subunit of NF-Y that is constitutively nuclear, NF-YA is found in both the nuclear and cytoplasmic compartments and accumulates in the nucleus in response to TGF-b1 treatment. Thus, our data (Figure 1 and Supplementary Figure 1) demonstrate that TGF-b drives colocalization of two components of NF-Y. NF-YC was also recently described to undergo nuclear relocalization during the cell cycle (Frontini et al., 2004). Unlike NF-YA, NF-YC accumulated in the nucleus following leptomycin B treatment, suggesting that the subcellular distributions of these two NF-Y subunits are controlled by distinct mechanisms. Surprisingly, these investigators found NF-YA to be solely nuclear in their experimental system. This likely reflects different experimental conditions, particularly those used for cell permeabilization, as in our hands too their conditions led to a loss of NF-YA cytoplasmic staining.

We observed no variation in the total level of NF-YA in TGF-b-treated cells. In agreement with this, serum stimulation of NIH3T3 cells blocked in G0 was recently shown to result in phosphorylation of NF-YA, while the total level of NF-YA remained constant (Yun et al., 2003). However, the actual level of NF-YA protein can change in certain situations, for instance increasing during monocyte to macrophage differentiation (Marziali et al., 1999) or decreasing in terminally differentiating myoblast C2C12 cells (Farina et al., 1999). We used cyclin A2 as a model gene to test the effect of the activation of NF-YA by TGF-b on transcription. Indeed, NF-Y associates with the promoters of several genes whose products help control the cell cycle. In addition, knockout experiments clearly showed the role of NF-Y in cell proliferation (Bhattacharya et al., 2003). However, recent studies suggest that NF-Y could regulate a wider spectrum of genes (Testa et al., 2005), opening the question of which subset of these genes might be targeted by TGF-b. One example might be procollagen A1 (COL1A1), as it was recently shown to be activated by TGF-b in cardiac fibroblasts through increased NF-Y binding (Lindahl et al., 2002). As expression of the TGF-b type II receptor is under the transcriptional control of a CCAAT box (Park et al., 2002;Huang et al., 2005), it is tempting to speculate that regulation of NF-Y activity by TGF-b could provide a feedback loop for TGF-b signaling.

We show that TGF-b1-dependent nuclear accumulation of NF-YA occurs in fibroblasts and epithelial cells. In both cell types, this requires activation of the ERK1/2 cascade. Although ERK1/2 are activated with similar kinetics by TGF-b1 in fibroblasts and in epithelial cells, the kinetics of NF-YA nuclear localization clearly differs between these two cell types. This difference correlates with the basal level of activation of the p38 cascade. Interestingly, exponentially growing epithelial MDCK or HEK cells (data not shown) contain barely detectable levels of basal p38 phosphorylation and show a much more rapid nuclear accumulation of NF-YA in response to TGF-b1. Taken together, these observations suggest that the activity levels of ERK and p38 help control the dynamics of NF-YA activation in response to TGF-b.

We do not know whether the effect of MAPK cascade activation on the dynamics of NF-YA nuclear relocation might be due to a phosphorylation of the NF-YA protein or of NF-YA regulating factors. Negative crosstalk between the ERK and p38 cascades has previously been described in keratinocytes (Efimova et al., 2003) and in the murine myeloid M1 cell lines, where p38 inhibition was shown to upregulate ERK activity (Hall and Davis, 2002). Regardless of the precise mechanism of regulation involved, our findings on NF-YA activation by TGF-b1 suggest that a single activation mechanism can sustain distinct dynamics of activation, depending on the cellular context. Thus, they provide novel insights on how TGF-b cytokines can generate distinct signaling patterns in different cell types.

Materials and methods

Cell lines, cell culture, transfections and reagents NIH3T3, MDCK and CCL39 cells were grown in DMEM supplemented with 10% fetal bovine serum (FBS). TGF-b1 was from RD Systems. For luciferase experiments, 10 5 CCL39 cells per 3.5 cm diameter Petri dish were transfected for 6 h with calcium phosphate in FBS 0.5% and further grown for 16 h with TGF-b1 (80 pM) in FBS 5%, prior to luciferase activity measurement (Promega). Luciferase experiments were performed in triplicate and data shown represent the average of three such experiments. Other transfections were achieved with Fugene (Roche) and TGF-b treatment was performed 24 h later. When mentioned, cycloheximide (10 mg/ml), PD98059, SB203580 (10 mM, Alexis Biochemicals), SP600125 (10 mM, Calbiochem) and okadaic acid (1 mM, Alexis Biochemicals) were added to the cell culture 30 min prior to TGF-b treatment and left thereafter.

Plasmids and oligonucleotides

Details of all constructs and oligonucleotides used are available upon request. mCCRE-CycA2-Luc is a pGL2-basic vector containing murine cyclin A2 promoter sequences (À177 to þ 100 relative to the most 3 0 transcription initiation site, mutated at the CCRE site). The mutation at the CCRE site prevents indirect regulating effects of TGF-b. The CCAAT box sequence was mut to ggccT by splice overlap extension PCR and constructs were sequenced.

Chromatin immunoprecipitation and fractionation

Chromatin immunoprecipitations (ChIP) were performed on NIH3T3 cells essentially as described (Fajas et al., 2001). Cells were incubated for various times with TGF-b1 (80 pM). Approximately 5 Â 10 6 cells were used per ChIP. An aliquot of each chromatin sample ('input') was taken prior to immunoprecipitation and purified in parallel to the immunoprecipitated DNA. Immunoprecipitations was carried out overnight with antibodies against NF-YA (sc-10779, Santa Cruz) or Tyk2 (#06-375, UBI). Immunoprecipitated and 'input' DNAs were assayed by both semiquantitative and real-time PCRs. For the semiquantitative PCR reactions, a 32 P-dCTP was incorporated during the amplification and aliquots of the PCR reactions were analysed in parallel with Maxam and Gilbert DNA sequence and analysed by PAGE and autoradiography. Quantitative PCR analysis was carried out with a light cycler apparatus (Roche) and for each locus analysed, both the immunoprecipitated and the input DNAs were quantified. PCR DNA fragments were circa 200-500 bp in length. Small scale chromatin fractionation was carried out as previously described (Wysocka et al., 2001).

Immunofluorescence NIH3T3 cells were grown on coverslips in six-well dishes (40 000 cells/well) in FCS 10%. After 48 h, they were treated with TGF-b1 (80pM) for various times. Cells were fixed with PFA (4%) for 30 min and ethanol permeabilized (EtOH 70%, overnight at 41C). Cells were incubated with the antibodies for 1 h at room temperature. Immunofluorescence was monitored by incubation with FITC and Cy-3 secondary antibodies. Coverslips were mounted with Permafluor (Immunon). The following primary antibodies were used: rabbit anti-NF-YB, monoclonal anti-NF-YA (Mab-7) (gifts from R Mantovani), monoclonal anti NF-YA (#556359, BD Pharmingen), Smad3 (sc-8332, Santa-Cruz), rabbit anti-Flag (#7425, Sigma), p44/42 and p38 MAPKs (#9102 and #9212,Cell Signaling). All immunofluorescence data are representative of at least three independent experiments.

Protein extracts and immunoblots

Protein extracts were prepared from actively growing subconfluent NIH3T3 fibroblasts treated with TGF-b1 (80 pM) for the indicated times. Cells were scraped in PBS containing NaF (50 mM) and vanadate (2 mM, Sigma). Nuclear extracts were prepared as described previously (Vignais et al., 1996). Buffers contained protease inhibitors (Roche) as well as microcystin (0.2 mM, Sigma-M2912) and vanadate (0.5 mM). The lysis buffer for whole cell extracts contained: 50 mM Tris (pH 8.0), 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 50 mM NaF, 25 mM NaPP, 1% NP-40, 1 mM DTT, 1 mM PMSF and 1 mM vanadate. Proteins were separated by SDS-PAGE (15%) and transferred to nitrocellulose membranes. After incubating with antibodies, blots were finally subjected to enhanced chemiluminescence substrate (Lumi-Light, Roche). The following primary antibodies were used: NF-YA monoclonal antibody (#556359, BD Pharmingen and gift from R Mantovani), Smad3 (sc-8332, Santa-Cruz), phospho-p44/42 MAPK (E10 and #9101S, Cell Signaling), p44/42 MAPK (#9102, Cell

Figure 1

 1 Figure 1 TGF-b treatment leads to nuclear accumulation of NF-YA in NIH3T3 cells. (a) NIH3T3 fibroblasts were treated for the indicated times (in min) with 80 pM TGF-b1. Endogenous NF-YA and Smad3 were visualized by immunofluorescence. Nuclei were stained with Hoechst. (b) Lysates were prepared from NIH3T3 cells incubated with TGF-b for the indicated times. Equal amounts of nuclear extracts (upper panel) and total extracts (lower panel) were analysed by immunoblotting with antibodies specific for NF-YA, Smad3, TBP and GAPDH.

Figure 2

 2 Figure 2 Effect of TGF-b treatment on NF-Y DNA binding and transcriptional activity. Chromatin immunoprecipitation (ChIP) was performed on NIH3T3 fibroblasts after TGF-b1 (80 pM) treatment. The chromatin samples were immunoprecipitated with antibodies specific for either NF-YA or Tyk2. The immunoprecipitated DNAs were analysed by both semiquantitative (a) and realtime (b) PCR. (a) PCR amplification of the CCAAT box region of the endogenous cyclin A promoter. The products of 32 cycles of amplification were detected using [a 32 P]dCTP and analysed by autoradiography of DNA-PAGE. 'Genomic DNA' is a control of native murine DNA and 'total DNA' corresponds to the chromatin input for the immunoprecipitations. (b) Percentage of immunoprecipitated DNA (ratio of immunoprecipitated versus input DNA) for each time point (in min) of TGF-b treatment. PCRs were performed using primer pairs amplifying the CCAAT box region or a region 700 bp upstream of the endogenous cyclin A2 promoter. Tyk2 indicates amplification of ChIPs performed at the 120 min time point with the Tyk2 antibody. The table indicates the increase in NF-YA binding to the endogenous cyclin A2 CCAAT box observed in response to TGF-b treatment. The increase in NF-YA binding upon TGF-b treatment was observed in several ChIP experiments. The diagram shows the data from two independent experiments. (c) ChIP analysis of TGF-b-induced binding of NF-YA to CCAAT boxes in the indicated promoters. The experiments were performed as described above. (d) The CCAAT box and NF-Y are required for TGF-b-dependent repression of cyclin A2 transcription. NIH3T3 cells were transfected with luciferase reporter genes driven by the cyclin A2 promoter bearing either the wt or a mut CCAAT box. Cells were then left untreated or induced with 80 pM TGF-b1. The left panel represents the relative luciferase units (RLU) measured in control (grey) or TGF-b-treated (black) cells. The right panel presents the degree of cyclin A2 trancriptional repression, measured as the ratio of luciferase activities in untreated versus TGF-b-treated cells.

Figure 3

 3 Figure 3 NF-YA nuclear accumulation in response to TGF-b requires neither protein neosynthesis nor Smad2/Smad3 signaling. NIH3T3 cells were seeded on coverslips and treated with 80 pM TGF-b for 90 min. The endogenous NF-YA and Smad3 were detected by immunofluorescence. (a) Cycloheximide (CHX, 10 mg/ml) was added to NIH3T3 cells 30 min prior to TGF-b treatment. (b) NIH3T3 cells were transfected with a vector encoding Flagtagged Smad7 protein 24 h prior to TGF-b treatment. Smad7 overexpression was monitored by immunofluorescence using a polyclonal Flag antibody.

Figure 4

 4 Figure 4 ERK1/2 activation is required for TGF-b-induced NF-YA nuclear accumulation. (a) Lysates were prepared from NIH3T3 cells treated with 100 pM TGF-b for the indicated times. Equal amounts of total extracts were analysed by western blotting with antibodies recognizing ERK, p38 and SAPK in both their unmodified and phosphorylated states. For a positive control, cells were treated for 15 min with 50 ng/ml anisomycin (ani). (b, c) NIH3T3 cells were pretreated with 10 mM PD98059, 10 mM SB203580, 10 mM SP600125 or 1 mM okadaic acid 30 min prior to a 60 min (b) or 90 min (c) induction with 100 pM TGF-b. (b) Whole cell extracts were analysed as in (a). (c) Endogenous NF-YA and Smad3 were visualized by immunofluorescence.

Figure 5

 5 Figure 5 MDCK epithelial cells and NIH3T3 fibroblasts show different kinetics of TGF-b-induced nuclear accumulation of NF-YA. (a) MDCKs were treated with 80 pM TGF-b1 for the indicated time in minutes. Endogenous NF-YA and Smad3 were visualized by immunofluorescence. (b) Lysates were prepared from MDCKs induced with TGF-b for the indicated times. Equal amounts of whole cell extracts were analysed by Western blotting with the indicated antibodies. (c) MDCKs were pretreated for 30 min with 10 mM PD98059 prior to a 15 min induction with TGF-b. Cells were treated as in (a).

Figure 6

 6 Figure 6 The TGF-b-dependent association of NF-YA with chromatin requires ERK activation. (a, b) MDCK cells were incubated with TGF-b for the indicated times after a 30 min pretreatment with PD98059 (PD) or with carrier only (Ctl). Nuclear extracts (a) and chromatin-enriched fractions (b) were prepared from these cells and analysed by immunoblotting with antibodies specific for Smad2/3, NF-YA and TBP. (c) The chromatin fraction of TGFb-treated cells (30 min time point) was treated with Mnase for the times indicated. The digested suspension was then centrifuged and the supernatant and pellet analysed by immunoblotting with anti NF-YA antibodies.

Figure 7

 7 Figure 7 Leptomycin B (LMB) treatment differentially affects ERK and NF-YA intracellular localization. NIH3T3 and MDCK cells were treated with 20 nM leptomycin B for 16 h. (a) Endogenous NF-YA and ERK were visualized by immunofluorescence. (b) Statistical analysis was carried out on two independent experiments for each cell line with either FITC-or cy3-conjugated antibodies for the detection of NF-YA. The left and right panels show the data for the endogenous NF-YA for respectively the NIH3T3 and the MDCK cells. The intracellular localization of an overexpressed GFP-tagged NF-YA protein was also investigated in the NIH3T3 cells. The data reflect the results of three independent transfection experiments (middle panel). The total number of cells counted for each condition is indicated (#). The histograms and the tables below show the percentage of cells harboring the different NF-YA intracellular localizations, in the control and LMB culture conditions.

Oncogene

Acknowledgements

We thank R Mantovani (University of Modene, Italy) for discussions and for providing antibodies and plasmids, K Matsumoto for providing plasmids. We also thank Annick Vie´for her excellent technical support. This work was funded by grants from l'Association pour la Recherche sur le Cancer (contracts 3304 to RAH, 4209 and 3446 to MLV) and La Ligue Contre le Cancer. SC was supported by the Ch Oberling Foundation.

Oncogene

Signaling), phospho-p38 and p38 MAPKs (#9211 and #9212, Cell Signaling), phospho-SAPK (#9251, Cell Signaling), SAPK (#9252, NewEngland Biolabs), monoclonal anti-TBP (clone 3G3). All blots shown are representative of at least 2 different experiments performed from different protein extracts.

Statistical analysis

Statistical analysis of the results was performed with the statistical package SAS system and with the ANOVA test. A P-value o0.05 was considered to be statistically significant.

Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc).