Cross-talk between the TGFβ and Wnt signaling pathways in murine embryonic maxillary mesenchymal cells

FEBS 29651

FEBS Letters 579 (2005) 3539–3546

Cross-talk between the TGFb and Wnt signaling pathways in murine embryonic maxillary mesenchymal cells Dennis R. Warner*, Robert M. Greene, M. Michele Pisano University of Louisville Birth Defects Center, Department of Molecular, Cellular, and Craniofacial Biology, 501 South Preston Street, Suite 301, Louisville, KY 40292, United States Received 8 March 2005; revised 11 May 2005; accepted 17 May 2005 Available online 2 June 2005 Edited by Lukas Huber

Abstract The transforming growth factor beta (TGFb) and Wnt signaling pathways play central roles regulating embryogenesis and maintaining adult tissue homeostasis. TGFb mediates its cellular effects through types I and II cell surface receptors coupled to the nucleocytoplasmic Smad proteins. Wnt signals via binding to a cell surface receptor, Frizzled, which in turn activates intracellular Dishevelled, ultimately leading to stabilization and nuclear translocation of b-catenin. Previous studies have demonstrated several points of cross-talk between the TGFb and Wnt signaling pathways. In yeast two-hybrid and GST-pull down assays, Dishevelled-1 and Smad 3 have been shown to physically interact through the C-terminal one-half of Dishevelled-1 and the MH2 domain of Smad 3. The current study demonstrates that co-treatment of murine embryonic maxillary mesenchyme (MEMM) cells with Wnt-3a and TGFb leads to enhanced reporter activity from TOPflash, a Wnt-responsive reporter plasmid. Transcriptional cooperation between TGFb and Wnt did not require the presence of a Smad binding element, nor did it occur when a TGFb-responsive reporter plasmid (p3TP-lux) was transfected. Overexpression of Smad 3 further enhanced the cooperation between Wnt and TGFb while overexpression of dominant-negative Smads 2 and 3 inhibited this effect. Co-stimulation with TGFb led to greater nuclear translocation of b-catenin, providing explanation for the effect of TGFb on Wnt-3a reporter activity. Wnt-3a exerted antiproliferative activity in MEMM cells, similar to that exerted by TGFb. In addition, Wnt-3a and TGFb in combination led to synergistic decreases in MEMM cell proliferation. These data demonstrate a functional interaction between the TGFb and Wnt signaling pathways and suggest that Wnt activation of the canonical pathway is an important mediator of MEMM cell growth.
2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: TGFb; Wnt; Orofacial; Cross-talk; Dishevelled; Smad; Transcription

* Corresponding author. Fax: +1 502 852 8309. E-mail address: [email protected] (D.R. Warner).

Abbreviations: TGFb, transforming growth factor beta; TbRI, type I TGFb receptor; TbRII, type II TGFb receptor; GSK-3b, glycogen synthase kinase 3b; Dvl, Dishevelled; APC, adenomatous polyposis coli; TCF, T-cell factor; LEF, lymphoid enhancer factor; MEMM, murine embryonic maxillary mesenchyme; GST, glutathione-S-transferase; FBS, fetal bovine serum; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate buffered saline

1. Introduction Embryonic development is under the influence of several major signaling pathways, including those initiated by members of the TGFb and Wnt families of secreted growth and differentiation factors. The TGFb superfamily is composed of three subfamilies: TGFbs, activins/inhibins, and bone morphogenetic proteins (BMPÕs) (for a recent review, see [1]). TGFb s elicit a plethora of biological responses ranging from morphogenesis during early embryonic development, to cellular proliferation, differentiation, apoptosis, and stimulation of extracellular matrix synthesis [2–6]. TGFb effects these processes by altering patterns of gene expression. The TGFb signaling cascade is initiated by binding of TGFb to a type II receptor-serine/threonine kinase (TbRII) that heterodimerizes with a type I receptor (TbRI, also a serine/threonine kinase), resulting in transphosphorylation of TbRI by TbRII [7]. Activated (phosphorylated) TbRI in turn phosphorylates the Smad proteins, transducers of the TGFb signaling cascade [8]. The Smads are categorized into three distinct groups based on function and sequence homology. R-Smads, substrates for TbRIs, are further divided into those regulated by TGFb/activin (Smads 2 and 3) and those regulated by BMPÕs (Smads 1, 5, and 8). Smad 4, a ‘‘common’’ Smad utilized by multiple TGFb superfamily members, heterodimerizes with phosphorylated R-Smads resulting in a complex that is imported into the nucleus [9]. Inhibitory Smads (Smads 6 and 7), attenuate sustained TGFb signaling by blocking receptor-activation of R-Smads [10,11]. Once in the nucleus, the coSmad/R-Smad complex binds to specific sequences within the promoters of TGFb-responsive genes and either stimulates or represses transcription. Transcriptional activation by Smads occurs with the aid of nuclear coactivators such as CBP and p300 [12], while transcriptional repression is effected by interaction with nuclear corepressors such as SnoN and c-Ski [13]. Thus, depending, in part, on the particular cellular complement of nuclear coactivators and corepressors, the transcriptional outcome of TGFb signaling will vary. R-Smads bind to DNA, albeit poorly, via their conserved MH1 domains. High affinity binding of Smads to DNA requires additional cofactors. For example, the proteins OAZ and FAST2 are required for Smad-dependent developmental processes by forming transcriptional complexes with Smads in the transcription of Xvent-2 and goosecoid, respectively [14,15]. Wnt was initially described in mice as the proto-oncogene int-1 [16] and later determined to be identical to Drosophila wingless [17] (Wnt is a contraction of wingless and int-1). The vertebrate Wnt gene family (currently 19 genes) encodes

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2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2005.05.024

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secreted, lipid modified glycoproteins that bind to serpentine receptors encoded by the Frizzled genes [18]. Wnts function as morphogens regulating early developmental processes such as axis specification, neural patterning, and organ development [19–22]. In addition, roles for Wnt in proliferation and apoptosis have been demonstrated experimentally in a number of cell types [23]. The central protein in the canonical Wnt signaling pathway is b-catenin, identified by its interaction with cadherins and its role in cell adhesion [24,25]. In the absence of Wnt, intracellular levels of free b-catenin are kept low through phosphorylation by glycogen synthase kinase 3b (GSK3b, [26,27]). Phosphorylation of b-catenin by GSK-3b is facilitated by two negative regulators: axin (conductin) and the tumor suppressor protein, adenomatous polyposis coli (APC) [28,29]. b-catenin, axin, APC, and GSK3b constitute a ‘‘destruction complex’’ [30]. Phospho-b-catenin binds to the beta transducin repeat-containing protein (bTrCP) component of this E3 ubiquitin ligase complex, which ubiquitinates b-catenin, marking it for elimination by the proteasome [31]. Wnt signaling antagonizes b-catenin phosphorylation and subsequent proteolysis by activating Dishevelled (Dvl) which disrupts formation of this destruction complex [32]. The mechanism of Wnt-Frizzled activation of Dvl involves its hyperphosphorylation and membrane translocation [33,34]. Inhibition of b-catenin phosphorylation leads to intracellular accumulation of the protein and concomitant translocation to the nucleus where it forms a complex with transcription factors of the T-cell factor/lymphoid enhancer factor (TCF/LEF) family that then binds to specific DNA sequences to activate gene transcription. Wnt also regulates planar cell polarity and convergent extension movements through a non-canonical pathway involving activation of Jun N-terminal kinase (JNK [35,36]). Wnts and TGFb superfamily members interact to regulate the transcription of a number of genes. For example, the TGFb superfamily member, Activin, and Wnt cooperate to stimulate expression of Siamois in Xenopus [37]. Transcription of Xtwn, also in Xenopus, requires a transcription complex that includes the TGFb-regulated Smad 2 and Smad 3, Smad 4, and TCF/ LEF [38]. Wnt and decapentaplegic signals are both required for expression of the Drosophila genes Vestigial and Ultrabithorax and Wnt and TGFb cooperate in formation of SpemannÕs organizer in Xenopus [39]. Antagonism between these two signaling pathways can occur through activation of TAK1 by TGFb, which inhibits canonical Wnt signaling in transfected HEK293 cells [40]. Finally, Axin, has been shown to enhance TbRI-dependent Smad phosphorylation [41]. In the experiments presented herein, functional cross-talk between the TGFb and Wnt signaling pathways in embryonic maxillary mesenchymal cells is demonstrated. Using transcription reporter assays combined with previous observations that Smads and Dvl physically interact [42], these data demonstrate several levels of cross-talk between TGFb and Wnt in cells derived from developing orofacial tissue.

2. Materials and methods 2.1. Animals and primary cell cultures ICR mice were obtained from Harlan (Indianapolis, IN), and housed at a temperature of 22 C with an alternating 12 h light/dark cycle and given access to food and water ad libitum. Mature male

D.R. Warner et al. / FEBS Letters 579 (2005) 3539–3546 and female mice were mated overnight and the presence of a vaginal plug the following morning was taken as evidence of mating and designated gestation day 0. Pregnant mice were euthanized on day 13 of gestation, a critical stage of murine orofacial development, embryos removed, embryonic maxillofacial tissue microdissected and placed in sterile, cold phosphate-buffered saline followed by gentle trypsinization, and plated at a density of 6 · 103 cells/cm2. Experiments utilizing primary cultures were initiated within 2 days of plating at which time the cells were approximately 50% confluent. These primary cultures are subsequently designated as murine embryonic maxillary mesenchyme (MEMM) cells. 2.2. Transfections and luciferase reporter assays For reporter assays, MEMM cells were seeded into 6-well tissue culture plates at a density of 7 · 104 cells/well and maintained in OptiMEM (GIBCO Invitrogen Corp., Gaithersburg, MD) supplemented with 5% heat-inactivated fetal bovine serum (Sigma, St. Louis, MO) at 37 C in 5% CO2/95% air. The following day, when plasmid transfections were performed, the cultures were 50–60% confluent. Cell cultures were transfected with plasmid DNAs (as below) using Effectene transfection reagent (Qiagen, Inc., Valencia, CA) according to the manufacturerÕs recommendations with the following ratios of reagents: Enhancer:DNA (8:1) and Effectene:DNA (15:1). For Wnt signaling reporter assays, the following amounts of plasmids were used, per well of a 6-well plate: 1 lg TOPflash or FOPflash (Upstate USA, Inc., Chicago, IL), 0.05 lg pRL-CMV (Promega, Madison, WI), 0.25 lg pCMV-Smad 3, or the dominant-negative mutants, pCMV-Smad 2 3S/A and pCMV-Smad 3 3S/A. The dominant-negative mutants of Smads 2 and 3 were created by replacing the C-terminal serine residues with alanine which prevents receptor-mediated phosphorylation [43]. The control plasmid pRL-CMV was co-transfected to normalize for transfection efficiencies within individual experiments. Sixteen hours post-transfection, cells were washed with fresh culture medium and stimulated with TGFb1 (2 ng/ml, R&D Systems Inc., Minneapolis, MN) for 24 h. Cells were harvested, lysates prepared, and luciferase activity determined with the Dual Luciferase Reporter Assay System (Promega) according to the method detailed by the manufacturer. Reporter luciferase activity (Firefly) was expressed relative to control luciferase activity (Renilla) for each sample. Each condition was assayed in triplicate and the experiment was performed twice with comparable results. 2.3. Cell proliferation assay MEMM cells were seeded into 24-well plates at a density of 3500 cells/cm2 and allowed to proliferate for 1–2 days in complete medium (Opti-MEM containing 5% FBS), at which time they were provided reduced serum media (Opti-MEM containing 0.25% FBS) and maintained for 48 h to achieve cell synchrony. Cell cultures were treated with control- or Wnt-3a-conditioned medium (50%, diluted in OptiMEM containing 5% FBS), 2 ng/ml TGFb1, or 25 mM LiCl and 2 lCi/ml [3H]thymidine (20 Ci/mmol, Perkin–Elmer Life Sciences, Boston, MA). Treatment with TGFb1 and LiCl was performed in the presence of 50% control-conditioned media in order to normalize media conditions so that a direct comparison of treatment efficacy could be made. At various time points, cultures were washed three times with 0.5 ml ice-cold PBS, twice for 10 min each with 0.5 ml ice-cold 10% (w/v) trichloroacetic acid (TCA), rinsed again three times with 0.5 ml ice-cold PBS and then solubilized for 30 min with 0.5 ml 1 N NaOH. Fifty ll glacial acetic acid was added to neutralize each sample and each was mixed with 10 ml Scintiverse scintillation cocktail (Fisher Chemicals, Fairlawn, NJ) and incorporation of [3H]thymidine into TCA-precipitable material was determined by liquid scintillation spectroscopy. 2.4. Preparation of Wnt-3a conditioned medium Mouse L-cells stably transfected with the cDNA for Wnt-3a were obtained from ATCC (Manassas, VA) and control, non-transfected L-cells were from Dr. Roel Nusse (Stanford University, Stanford, CA). Wnt-3a- and control-conditioned media were prepared according to the method of Shibamoto and were stored for up to one year without appreciable loss of activity [44]. Using this method, the concentration of soluble Wnt-3a in 100% conditioned media is approximately 400 ng/ml [44], however, there were batch-to-batch variations in activity.

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2.5. Construction of dominant-negative Smad 2 and Smad 3 To create dominant-negative mutants of both Smad 2 and Smad 3, PCR was utilized using wild-type Smad 2 and Smad 3 cDNA as a template in order to change the three carboxyl terminal serine residues to alanine (Smad 2, -SSMS to -AAMA; Smad 3, -SSVS to -AAVA). This removed the phosphorylation site necessary for activation [43]. Both mutants were cloned into pCMV-myc (BD Biosciences) resulting in fusion of a c-myc epitope to the amino terminus. 2.6. Nuclear translocation of b-catenin To determine cytosolic and nuclear levels of b-catenin in MEMM cells, primary cultures were established in 10 cm tissue culture plates (2 plates per treatment) and grown to 70% confluence. Cultures were serum-deprived for 16 h in DME/F12 (Sigma), and then stimulated with control- or Wnt-3a-conditioned media in the presence or absence of 2 ng/ml TGFb1 for 3 h. Cells were washed twice with ice-cold PBS and scraped in 1 ml PBS. Cells were collected by centrifugation at 2000 · g for 2 min and cytosolic and nuclear protein fraction prepared using the NE-PER nuclear and cytoplasmic extraction kit from Pierce (Rockford, IL) according to the manufacturerÕs instructions. Five10 lg were analyzed by SDS–PAGE and immunoblotting with antiactive b-catenin antibody (clone 8E4; Upstate Cell Signaling Solutions, Lake Placid, NY), which recognizes non-phosphorylated b-catenin. Quantitative assessment of translocation was determined by densitometry using NIH Image 1.63f. 2.7. Statistical analyses Data from reporter and proliferation assays was analyzed using one-way ANOVA followed by BonferroniÕs multiple comparison test. P-values less than or equal to 0.05 were considered significant.

Fig. 1. Activation of the canonical Wnt signaling pathway in MEMM cells. Primary cultures of MEMM cells were established and seeded into 6-well plates at a density of 7.5 · 104 cells/well. Cells were allowed to reach 50–60% confluence and were transfected with 1 lg of the TCF-reporter plasmid, TOPflash, or a mutated version, FOPflash, which does not respond to Wnt signaling, and 50 ng pRL-CMV, which served as a control to ascertain transfection efficiency. Twenty-four hours post transfection, cells were treated with control- or Wnt-3aconditioned media (50%, diluted in 5% FBS/Opti-MEM) for an additional 24 h. Cell lysates were prepared and assayed for Firefly luciferase (expressed from TOPflash or FOPflash) or Renilla luciferase activity (from pRL-CMV). Data are expressed as the ratio of Firefly luciferase activity to that of Renilla luciferase. Each data point represents the means ± standard deviation of triplicate determinations. Wnt-3a conditioned medium stimulated transcription specifically from TOPflash (P < 0.05, one-way ANOVA and BonferroniÕs test), with no effect on FOPflash.

3. Results 3.1. Stimulation of a Wnt reporter construct in MEMM cells In order to determine the ability of MEMM cells to respond to signals through the canonical Wnt signaling pathway (i.e. through b-catenin and TCF/LEF), primary cultures of MEMM cells were transfected with the reporter construct, TOPflash. This construct contains six copies of an optimized TCF-consensus binding site upstream of Firefly luciferase. FOPflash, which contains mutations in each of the six TCF consensus sites rendering it insensitive to Wnt signaling, was transfected into MEMM cells as a negative control [45]. Twenty-four hours following transfection, cells were treated for an additional 24 h with control- or Wnt-3a-conditioned medium (50% concentration) and luciferase activity measured. Wnt-3a was chosen because it has been shown to activate the canonical Wnt pathway through stabilization of b-catenin [44]. Additionally, Wnt-3a was found to be present in the developing secondary palate (manuscript in preparation). As shown in Fig. 1, Wnt-3a effectively stimulated transcription of luciferase from the TOPflash reporter construct, but not from FOPflash, demonstrating that MEMM cells contain a functional canonical Wnt signaling pathway. 3.2. Cross-talk between the Wnt-3a and TGFb signaling pathways in MEMM cells In order to define a functional interaction between the Wnt3a and TGFb signaling pathways in MEMM cells, cultures were transfected with TOPflash, FOPflash, or p3TP-lux, a luciferase reporter plasmid highly responsive to Smad-mediated TGFb signaling [46] and stimulated with 50% Wnt-3a conditioned medium and/or 2 ng/ml TGFb. The results shown in Fig. 2 demonstrate that Wnt-3a stimulation led to activation of TOPflash but not FOPflash, which is consistent with the re-

Fig. 2. TGFb and Wnt cooperate to stimulation transcription from a reporter plasmid containing TCF binding sites. Primary cultures of MEMM cells were transfected with TOPflash, FOPflash, or the p3TPlux reporter plasmids and pRL-CMV. Cultures were incubated for 24 h and then stimulated with 50% conditioned medium (control or Wnt-3a) in the presence or absence of 2 ng/ml TGFb1 for 24 h. Induced-expression of luciferase was measured as detailed in Section 2 and is reported as the means ± standard deviation of triplicate determinations. Wnt-3a treatment resulted in an increase in transcription from TOPflash, but not FOPflash or p3TP-lux. TGFb treatment resulted in transcription only from p3TP-lux. Co-treatment of MEMM cells with both Wnt-3a and TGFb led to a synergistic response from TOPflash (P < 0.05, as compared to Wnt-3a alone, one-way ANOVA and BonferroniÕs test) but not p3TP-lux.

sults presented in Fig. 1. However, co-treatment with both 50% Wnt-3a conditioned medium and 2 ng/ml TGFb led to an enhanced activation of TOPflash (P < 0.05 compared to

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Wnt-3a alone) (Fig. 2). The increase appeared to be specific to the TCF reporter plasmid, as no effect of Wnt-3a on transcription from p3TP-lux was detected either in the presence or absence of TGFb. TGFb alone had no affect on luciferase transcription from TOPflash or FOPflash, nor did Wnt-3a alone have any affect on transcription from p3TP-lux. To further examine cross-talk between the TGFb and Wnt-3a signaling pathways in MEMM cells, and to determine if this interaction is Smad-dependent, Smad 3 was overexpressed in MEMM cells also transfected with TOPflash. Cells were then stimulated with Wnt-3a conditioned medium with or without TGFb. Overexpression of Smad 3 led to a generalized increase in basal TOPflash reporter activity (Fig. 3). However, the effect is not specific to TOPflash, as basal activities from FOPflash are also elevated. Smad 3 overexpression led to an increase in transcriptional activity from TOPflash in response to Wnt3a, from 1.5-fold to 1.9-fold, compared to control values (Fig. 3). Furthermore, the transcriptional activation observed with Wnt-3a and TGFb was enhanced in the presence of excess Smad 3 (7-fold stimulation over basal, compared to 2-fold when Smad was not overexpressed, P < 0.01 vs. Wnt-3a alone), suggesting that Smads can mediate cross-talk between TGFb and Wnt. Interestingly, overexpression of Smad 3 also led to increases in transcription of TOPflash by TGFb, to a level actually greater than that stimulated by Wnt-3a alone. Although Smad 3 led to increased basal activity from FOPflash, no specific effect of Wnt-3a conditioned media or TGFb was observed on luciferase levels expressed from this plasmid. To further examine the Smad dependence on the synergistic activation of TOPflash by Wnt-3a and TGFb, dominant-negative mutants of both Smad 2 and Smad 3 were overexpressed in TOPflash- or FOPflash-transfected MEMM cells and then the cultures were stimulated with Wnt-3a conditioned media in the presence or absence of TGFb (Fig. 4). These domi-

Fig. 3. Overexpression of Smad 3 in MEMM cells leads to enhanced Wnt-3a-TGFb-mediated transcription. Primary cultures of MEMM cells were transfected with the TOPflash or FOPflash reporter construct, pRL-CMV, and the expression vector pCMV-myc without a cDNA insert (empty vector) or with full-length Smad 3 followed by treatment with 50% conditioned medium (control or Wnt-3a) with or without 2 ng/ml TGFb1 for 24 h. The inset is a Western blot with antimyc antibodies demonstrating expression of myc-Smad 3. Overexpression of Smad 3 led to a marked increase in both basal and stimulated transcription of TOPflash and the cooperation between TGF and Wnt-3a in stimulating transcription was enhanced. Although overexpression of Smad 3 led to increased activity from FOPflash, there was no effect from treatment with Wnt-3a conditioned media either in the presence or absence of TGFb.

D.R. Warner et al. / FEBS Letters 579 (2005) 3539–3546

Fig. 4. Inhibition of TGFb signaling through overexpression of dominant-negative mutants of Smads 2 and 3 blocks cooperation between TGFb and Wnt-3a in MEMM cells. Primary cultures of MEMM cells were transfected with TOPflash or FOPflash, pRLCMV, and either pCMV-myc (empty vector) or pCMV-myc vectors that contained dominant-negative mutants of Smad 2 and Smad 3 (Dom.-neg.). Transfected cell cultures were treated with control- or Wnt-3a-conditioned medium in the presence or absence of 2 ng/ml TGFb1, and luciferase activity measured and normalized to that from pRL-CMV. The inset is a Western blot with anti-myc antibodies demonstrating expression of both myc-Smad 2 3S/A and myc-Smad 3 3S/A. Inhibition of TGFb signaling with dominant-negative mutants of Smads 2 and 3 not only led to an increased ability of Wnt-3a to induce transcription from TOPflash, but also blocked the ability of TGFb to enhance Wnt-3a-mediated transcription. Transfection with dominant-negative mutants of Smad 2 and Smad 3 led to a general increase in activity from FOPflash, but there was no specific activation from Wnt-3a or TGFb stimulation.

nant-negative mutants of Smad 2 and 3 contain mutations of the carboxyl-terminal serine residues that prevent TGFb receptor-mediated phosphorylation and effectively block signaling through the TGFb pathway [43]. Inhibition of TGFb signaling by dominant-negative mutants of Smad 2 and Smad 3 blocked the combined activation of TOPflash by Wnt-3a and TGFb (Fig. 4). Similar to the results with overexpression of wild-type Smad 3 (Fig. 3), overexpression of dominant-negative Smads 2 and 3 resulted in an enhanced ability of Wnt-3a to increase transcription on TOPflash (from 1.6-fold to 3.5-fold). The dominant-negative Smad 2 and Smad 3 mutants blocked the low level of TGFb-mediated stimulation of TOPflash reporter assay observed in this experiment. Although mediating a small increase in basal activity, Smad 2 3S/A and Smad 3 3S/A had no effect on FOPflash reporter activity. These data suggest that Smads may act as a cofactor in the Wnt signaling pathway and that Smad phosphorylation may not be necessary for enhancement of Wnt signaling, however it is required for TGFb-mediated effects on TOPflash transcription. 3.3. Cross-talk between TGFb and Wnt downstream of Frizzled and Dvl To determine if the effect of TGFb on Wnt-3a-transcriptional activity requires signaling through the Frizzled receptors and Dvl, MEMM cells were transfected with TOPflash or FOPflash and stimulated with 25 mM LiCl in the presence or absence of 2 ng/ml TGFb. Lithium ions directly inhibit GSK-3b and stimulate the canonical pathway by increasing b-catenin levels independent of receptor activation [47]. LiCl was effective in stimulating luciferase activity from TOPflash (P < 0.001 vs. NaCl control) (Fig. 5), with a response higher

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3543 Table 1 Nuclear translocation of b-catenin in MEMM cells

Fig. 5. Synergistic transcriptional activation of TOPflash by TGFb and LiCl. Primary cultures of MEMM cells were transfected with TOPflash or FOPflash, and pRL-CMV. Twenty-four hours post transfection, cells were treated with either control- or Wnt-3aconditioned medium, 25 mM NaCl or 25 mM LiCl in the presence or absence of 2 ng/ml TGFb1 for 24 h. Each data point represents the means ± standard deviation of the luciferase activity from triplicate transfections. LiCl efficiently stimulated transcription of TOPflash (P < 0.001, compared to NaCl), and when combined with TGFb, lead to a marked increase in luciferase transcription (P < 0.001, compared to LiCl alone). For comparison, Wnt-3a ± TGFb was also tested and was consistent with earlier observations (not shown). No effect was observed with FOPflash.

than that obtained with Wnt-3a conditioned media (not shown). It is unclear why stimulation of MEMM cells with Wnt-3a results in a lower response than with LiCl, however, the receptor expression (Frizzled) and concentration of Wnt3a in the conditioned media may combine to result in lower signals. Nevertheless, simultaneous treatment with LiCl and TGFb, resulted in a greater level of luciferase activity than that observed with LiCl alone (P < 0.001). These data demonstrate that the cooperation between TGFb and Wnt-3a is likely mediated downstream of Frizzled and Dvl. 3.4. TGFb co-stimulation with Wnt-3a leads to increased b-catenin nuclear translocation One explanation for the positive effect of TGFb on Wnt-3amediated TOPflash reporter activity could be due to enhanced nuclear translocation of b-catenin. When MEMM cells were stimulated with Wnt-3a conditioned media, increased nuclear levels of b-catenin were detected with an antibody specific for non-phosphorylated (i.e. activated) b-catenin (Table 1). Little effect by TGFb alone on b-catenin levels was detected. In the presence of 2 ng/ml TGFb, stimulation with Wnt-3a conditioned media led to a small, but reproducible (1.5–1.8fold) increase in the level of activated b-catenin found in the nucleus. As b-catenin is a transcriptional co-factor, these relatively small changes in nuclear levels may lead to much larger responses seen at the transcriptional level. Since Smads have been shown to bind b-catenin [48], it is possible that a Smadb:catenin complex formed by stimulation with both TGFb and Wnt-3a leads to the increase in nuclear b-catenin observed in this experiment. 3.5. Cross-talk between TGFb and Wnt-3a in regulation of MEMM cell proliferation TGFb and Wnt are both able to alter cell proliferation [49– 51]. TGFb negatively regulates the proliferation of MEMM cells [52]. To examine the effect of Wnt-3a on MEMM cell proliferation, cells were synchronized by serum deprivation and

Treatment

Densitometric units Exp. 1

Exp. 2

Control TGFb Wnt-3a Wnt-3a + TGFb

163 185 219 289

419 359 506 636

Average fold-change

– No-change 1.2 ± 0 1.6 ± 0.1

Primary cultures of MEMM cells were serum-starved for 16 h and stimulated with control- or Wnt-3a conditioned media ±2 ng/ml TGFb1 for 3 h. Cells were harvested and total nuclear protein was isolated as described in Section 2. Nuclear protein (5 lg) was analyzed by SDS–PAGE and Western blotting with anti-b-catenin (active, nonphosphorylated). The level of b-catenin present in the nucleus was determined by densitometry with NIH image 1.63 and data are presented from two independent experiments. The data are also expressed as the average fold-change ± range, relative to control values.

the effect of various treatments on cell proliferation was ascertained by measuring [3H]thymidine incorporation into DNA. As has been previously demonstrated, TGFb exerts an antiproliferative effect on MEMM cells [52] (Fig. 6). Treatment with Wnt-3a conditioned medium inhibited MEMM cell proliferation to a similar extent as that seen in response to 2 ng/ ml TGFb. Co-treatment of MEMM cells with both Wnt-3a conditioned medium and TGFb resulted in a further reduction in the rate of proliferation of MEMM cells (P < 0.05 compared to Wnt-3a or TGFb alone; Mann–Whitney test). To determine if the effect observed with Wnt-3a is mediated through the canonical pathway, the effect of 25 mM LiCl on cell proliferation was examined (Fig. 6). Although 25 mM LiCl inhibited cell proliferation, it was not as effective as Wnt-3a conditioned medium in inhibiting MEMM cell proliferation. Whether this indicates that Wnt-3a utilizes alternate pathways,

Fig. 6. TGFb and Wnt-3a cooperate to inhibit MEMM cell proliferation. Primary cultures of MEMM cells were seeded into 24-well plates at a density of 7000 cells/well. One day after seeding, medium was replaced with reduced-serum medium (0.25% FBS) and incubated for an additional 48 h to synchronized the cells. Control- or Wnt-3aconditioned medium (50%, diluted in 5% FBS/Opti-MEM) containing the TGFb1 vehicle (0.1% BSA, 4 mM HCl), 2 ng/ml TGFb1, 25 mM NaCl, or 25 mM LiCl was then added to the cells. In addition, all medium contained 2 lCi/ml [3H]thymidine (20 Ci/mmol). At the indicated time following addition of TGFb or Wnt-3a, the cells were harvested and the amount of TCA-insoluble [3H]thymidine was determined and expressed as cpm ± standard deviation of four samples. TGFb and Wnt-3a inhibited MEMM cell proliferation (P < 0.001, compared to control at 24 and 48 h). The combination of Wnt-3a and TGFb led to a small synergistic decrease in MEMM cell proliferation (P < 0.05).

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in addition to the canonical pathway to inhibit cell proliferation, or that LiCl is not as efficacious as Wnt-3a is unclear. LiCl may also have other cellular effects, such as increasing inositol lipids, which may influence its efficacy in this assay [53]. These data do suggest, however, that Wnt-3a can inhibit proliferation of MEMM cells, in part, through control of b-catenin levels. The cooperation between TGFb and Wnt-3a in regulating MEMM cell proliferation may not extend to other cellular phenomena as no cooperation was observed in TGFb-induced up-regulation of gelatinase B (data not shown). This latter observation is consistent with reporter assays demonstrating no effect of Wnt-3a conditioned medium on transcription from the TGFb-responsive reporter, p3TP-Lux (Fig. 2).

4. Discussion TGFb and Wnt activate two well-characterized signaling pathways that function during embryonic development and in adult tissue homeostasis. The TGFbs and Wnts bind to cell surface receptors that elicit nuclear translocation of a transcription factor that associates with additional nuclear proteins to promote specific changes in gene expression. Cell-specific interpretation of extracellular signals depends, in part, on integration between distinct signaling pathways. Signaling pathway crosstalk is now recognized as a critical mechanism by which inputs from multiple pathways can cooperate in gene transcription, or the means by which one signaling pathway can silence a competing or antagonistic pathway. Multiple levels of cooperation between the TGFb and Wnt signaling pathways in regulating gene expression have been documented. For example, Wnt and TGFb cooperate to induce the expression of Vestigial and Ultrabithorax [54,55]. Labbe´ et al. [56] demonstrated that Smads 2, 3, and 4 bind to LEF1, to synergistically activate the Xtwn promoter in Xenopus. Previously, it was demonstrated that Smad 3 binds to Dvl-1 in a region encompassing most of the PDZ domain and the entire DEP domain [42]. The data presented in this report is the first demonstration that MEMM cells respond to Wnt by increasing transcription through the canonical pathway. Activation of the cell polarity pathway (through activation of JNK) was also tested using JNK activation-specific antibodies, however, no activation of JNK was observed in response to Wnt-3a or Wnt-5a (data not shown). Due to the difficulties in obtaining functional Wnts, others were not tested for their ability to stimulate the canonical and cell polarity pathways in MEMM cells. TGFb alone activated TOPflash in some experiments (e.g. Figs. 3 and 4), which is consistent with previous reports demonstrating that TGFb is capable of activating this reporter construct in other cell types [57]. This effect was dependent upon Smads and activation of the TGFb pathway. However, overexpression of Smad alone (either wild-type or dominantnegative mutants), even in the absence of TGFb was sufficient to render MEMM cells more responsive to Wnt. These data suggest that Smads have the ability to serve as a cofactor in the Wnt signaling pathway but may not require activation of the TGFb pathway. The inability of Wnt-3a to elicit a transcriptional response from p3TP-lux suggests that Wnt-3a cannot stimulate transcription from a promoter consisting of only a Smad binding element. This is consistent with the observa-

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tion that Wnt had no effect on the expression of gelatinase B, a TGFb-responsive gene, in MEMM cells, either in the presence or absence of TGFb (data not shown). The observed cooperation between TGFb and Wnt in MEMM cells on transcription of TOPflash is not consistent with an earlier report examining the functional interaction between these two pathways. Labbe´ et al. demonstrated that TGFb and Wnt-3a cooperate in the regulation of the Xenopus homeobox gene Xtwn, but demonstrated that binding sites for both Smads and TCF are required, not only in the natural promoter, but also in artificial plasmid constructs (e.g. TOPflash, [56]). However, in this case the Wnt signaling pathway was stimulated by overexpression of LEF1 in a cell line containing activating mutations of b-catenin (HepG2) and may not reflect endogenous activation by the natural ligand, Wnt. A recent report demonstrated that treatment of mesenchymal progenitor cells with TGFb resulted in transcriptional activation of TOPflash and nuclear translocation of b-catenin in the absence of exogenous Wnt signaling [57]. These data suggest that responses to TGFb, Wnt, and combined TGFb-Wnt treatment may be tissue-specific since no consistent effect of TGFb on TOPflash was found in MEMM or Mv1Lu cells. Since nuclear translocation of b-catenin leads to activation of transcription, any changes in either its degradation or nuclear import would lead to alterations in transcription. Co-treatment of MEMM cells with TGFb and Wnt-3a conditioned media led to increased levels of b-catenin found in the nucleus, and may provide one mechanism for the greater level of Wnt3a-mediated TOPflash reporter activity conferred by TGFb. Although the increases seen when TGFb was included with Wnt-3a were small, relatively minor changes in levels of transcription factors could potentially lead to larger transcriptional changes. Whether Smads and b-catenin form a complex that is more efficiently imported into the nucleus, or whether Smads and b-catenin independently move into the nucleus and form a more effective transcriptional complex awaits further investigation. As the ultimate cellular effect and mechanisms of signaling by both TGFb and Wnt-3a are complex, other mechanisms mediating cross-talk between these two pathways may be operational and thus the simple increase in nuclear b-catenin levels may be insufficient to explain this phenomenon. The increase in b-catenin, however, may explain in part the increase in TOPflash reporter activity seen when MEMM cells are treated with both TGFb and Wnt-3a. The data presented in the current report supports a model whereby Smad is a common cofactor for both the TGFb and Wnt signaling pathways. It is possible that the observed interaction between TGFb and Wnt is mediated by CREB binding protein (CBP), since CBP, through its interaction with Smads [12], TCF [58], and b-catenin [59] is a common transcriptional co-activator for both TGFb and Wnt. Although the role(s) of Wnts in orofacial development remain largely unknown, the above data suggest that Wnts, in particular those stimulating the canonical pathway, are important mediators of cell proliferation since Wnt-3a exerted a strong antiproliferative response on MEMM cells. This may be particularly significant for development of the secondary palate, a primary research focus in this laboratory, since alterations in mesenchyme cell proliferation may lead to clefts of the secondary palate [60,61]. In addition, synergistic cross-talk between Wnt-3a and TGFb on promoters containing TCF binding sites may al-

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ter gene transcription profiles (i.e. some genes may require inputs from both Wnt and TGFb) or TGFb may modulate the strength of the Wnt-b-catenin/TCF signal. Although the biological effects of Wnt and TGFb, and cross-talk between the two, has been defined in a cell culture system, the ultimate goal is to understand their roles in embryogenesis. Thus, data from the experiments presented in the present report using cultured embryonic mesenchyme cells can be used to design experimental approaches to elucidate signaling mechanisms in craniofacial development in vivo. Acknowledgements: The authors thank Dr. Roel Nusse (Stanford University) for mouse L-cells and Dr. Joan Massague´ (Memorial SloanKettering Cancer Center) for p3TP-lux. This work was supported, in part, by NIH grants DE12363 (to M.M.P.), DE12858, DE05550, and P20 RR017702 from the COBRE Program of the National Center for Research Resources (all to R.M.G.), and the Commonwealth of Kentucky Research Challenge Trust Fund.

3545

[16] [17]

[18]

[19] [20] [21] [22]

References [23] [1] Shi, Y. and Massague´, J. (2003) Mechanisms of TGF-beta signaling from cell membrane to the nucleus. Cell 113, 685–700. [2] Zorn, A.M., Butler, K. and Gurdon, J.B. (1999) Anterior endomesoderm specification in Xenopus by Wnt/beta-catenin and TGF-beta signalling pathways. Dev. Biol. 209, 282–297. [3] Edwards, D.R., Murphy, G., Reynolds, J.J., Whitham, S.E., Docherty, A.J.P., Angel, P. and Heath, J.K. (1987) Transforming growth factor beta modulates the expression of collagenase and metalloproteinase inhibitor. EMBO J. 61, 1899–1904. [4] Roberts, A.B., Anzano, M.A., Wakefield, L.M., Roche, N.S., Stern, D.F. and Sporn, M.B. (1985) Type beta transforming growth factor: a bifunctional regulator of cellular growth. Proc. Natl. Acad. Sci. USA 82, 119–123. [5] Rotello, R.J., Lieberman, R.C., Purchio, A.F. and Gerschenson, L.E. (1991) Coordinated regulation of apoptosis and cell proliferation by transforming growth factor beta 1 in cultured uterine epithelial cells. Proc. Natl. Acad. Sci. USA 88, 3412–3415. [6] Ignotz, R.A. and Massague, J. (1985) Type beta transforming growth factor controls the adipogenic differentiation of 3T3 fibroblasts. Proc. Natl. Acad. Sci. USA 82, 8530–8534. [7] Ventura, F., Doody, J., Liu, F., Wrana, J.L. and Massague´, J. (1994) Reconstitution and transphosphorylation of TGF-beta receptor complexes. EMBO J. 13, 5581–5589. [8] Kretzschmar, M., Liu, F., Hata, A., Doody, J. and Massague´, J. (1997) The TGF-beta family mediator Smad1 is phosphorylated directly and activated functionally by the BMP receptor kinase. Genes Dev. 11, 984–995. [9] Lagna, G., Hata, A., Hemmati-Brivanlou, A. and Massague´, J. (1996) Partnership between DPC4 and SMAD proteins in TGFbeta signalling pathways. Nature 383, 832–836. [10] Imamura, T., Takase, M., Nishihara, A., Oeda, E., Hanai, J., Kawabata, M. and Miyazono, K. (1997) Smad6 inhibits signalling by the TGF-beta superfamily. Nature 389, 549–551. [11] Souchelnytskyi, S., Nakayama, T., Nakao, A., More´n, A., Heldin, C.H., Christian, J.L. and ten Dijke, P. (1998) Physical and functional interaction of murine and Xenopus Smad7 with bone morphogenetic protein receptors and transforming growth factor-receptors. J. Biol. Chem. 273, 25364–25370. [12] Pouponnot, C., Jayaraman, L. and Massague´, J. (1998) Physical and functional interaction of SMADs and p300/CBP. J. Biol. Chem. 273, 22865–22868. [13] Stroschein, S.L., Wang, W., Zhou, S., Zhou, Q. and Luo, K. (1999) Negative feedback regulation of TGF-beta signaling by the SnoN oncoprotein. Science 286, 771–774. [14] Hata, A., Seoane, J., Lagna, G., Montalvo, E., HemmatiBrivanlou, A. and Massague´, J. (2000) OAZ uses distinct DNAand protein-binding zinc fingers in separate BMP-Smad and Olf signaling pathways. Cell 100, 229–240. [15] Labbe, E., Silvestri, C., Hoodless, P.A., Wrana, J.L. and Attisano, L. (1998) Smad 2 and Smad 3 positively and negatively

[24]

[25]

[26] [27] [28]

[29]

[30]

[31] [32]

[33]

[34] [35]

regulate TGFb-dependent transcription through the forkhead DNA-binding protein FAST2. Mol. Cell 2, 109–120. Nusse, R. and Varmus, H.E. (1982) Many tumors induced by the mouse mammary tumor virus contain a provirus integrated in the same region of the host genome. Cell 31, 99–109. Rijsewijk, F., Schuermann, M., Wagenaar, E., Parren, P., Weigel, D. and Nusse, R. (1987) The Drosophila homolog of the mouse mammary oncogene int-1 is identical to the segment polarity gene wingless. Cell 50, 649–657. Bhanot, P., Brink, M., Samos, C.H., Hsieh, J.C., Wang, Y., Macke, J.P., Andrew, D., Nathans, J. and Nusse, R. (1996) A new member of the Frizzled family from Drosophila functions as a Wingless receptor. Nature 382, 225–230. Zecca, M., Basler, K. and Struhl, G. (1996) Direct and long-range action of a wingless morphogen gradient. Cell 87, 833–844. Parr, B.A. and McMahon, A.P. (1995) Dorsalizing signal Wnt-7a required for normal polarity of D-V and A-P axes of mouse limb. Nature 374, 350–353. Ikeya, M., Lee, S.M., Johnson, J.E., McMahon, A.P. and Takada, S. (1997) Wnt signalling required for expansion of neural crest and CNS progenitors. Nature 389, 966–970. Pandur, P., Lasche, M., Eisenberg, L.M. and Kuhl, M. (2002) Wnt-11 activation of a non-canonical Wnt signalling pathway is required for cardiogenesis. Nature, 636–641. Chen, S., Guttridge, D.C., You, Z., Zhang, Z., Fribley, A., Mayo, M.W., Kitajewski, J. and Wang, C.Y. (2001) Wnt-1 signaling inhibits apoptosis by activating beta-catenin/T cell factor-mediated transcription. J. Cell Biol. 152, 87–96. Ozawa, M., Baribault, H. and Kemler, R. (1989) The cytoplasmic domain of the cell adhesion molecule uvomorulin associates with three independent proteins structurally related in different species. EMBO J. 8, 1711–1717. Nagafuchi, A. and Takeichi, M. (1989) Transmembrane control of cadherin-mediated cell adhesion: a 94 kDa protein functionally associated with a specific region of the cytoplasmic domain of Ecadherin. Cell Regul. 1, 37–44. Peifer, M., Pai, L.M. and Casey, M. (1994) Phosphorylation of the Drosophila adherens junction protein Armadillo: roles for wingless signal and zeste-white 3 kinase. Dev. Biol. 166, 543–556. van Noort, M., Meeldijk, J., van der Zee, R., Destree, O. and Clevers, H. (2002) Wnt signaling controls the phosphorylation status of beta-catenin. J. Biol. Chem. 277, 17901–17905. Ikeda, S., Kishida, S., Yamamoto, H., Murai, H., Koyama, S. and Kikuchi, A. (1998) Axin, a negative regulator of the Wnt signaling pathway, forms a complex with GSK-3beta and betacatenin and promotes GSK-3beta-dependent phosphorylation of beta-catenin. EMBO J. 17, 1371–1384. Rubinfeld, B., Albert, I., Porfiri, E., Fiol, C., Munemitsu, S. and Polakis, P. (1996) Binding of GSK3beta to the APC-beta-catenin complex and regulation of complex assembly. Science 272, 1023– 1026. Kishida, S., Yamamoto, H., Ikeda, S., Kishida, M., Sakamoto, I., Koyama, S. and Kikuchi, A. (1998) Axin, a negative regulator of the wnt signaling pathway, directly interacts with adenomatous polyposis coli and regulates the stabilization of beta-catenin. J. Biol. Chem. 273, 10823–10826. Aberle, H., Bauer, A., Stappert, J., Kispert, A. and Kemler, R. (1997) beta-catenin is a target for the ubiquitin-proteasome pathway. EMBO J., 3797–3804. Li, L., Yuan, H., Weaver, C.D., Mao, J., Farr, G.H., Sussman 3rd, D.J., Jonkers, J., Kimelman, D. and Wu, D. (1999) Axin and Frat1 interact with Dvl and GSK, bridging Dvl to GSK in Wntmediated regulation of LEF-1. EMBO J. 18, 4233–4240. Axelrod, J.D., Miller, J.R., Shulman, J.M., Moon, R.T. and Perrimon, N. (1998) Differential recruitment of Dishevelled provides signaling specificity in the planar cell polarity and Wingless signaling pathways. Genes Dev. 12, 2610–2622. Lee, J.S., Ishimoto, A. and Yanagawa, S. (1999) Characterization of mouse dishevelled (Dvl) proteins in Wnt/Wingless signaling pathway. J. Biol. Chem. 274, 21464–21470. Yamanaka, H., Moriguchi, T., Masuyama, N., Kusakabe, M., Hanafusa, H., Takada, R., Takada, S. and Nishida, E. (2002) JNK functions in the non-canonical Wnt pathway to regulate convergent extension movements in vertebrates. EMBO J. 3, 69– 75.

3546 [36] Boutros, M., Paricio, N., Strutt, D. and Mlodzik, M. (1998) Dishevelled activates JNK and discriminates between JNK pathways in planar polarity and wingless signaling. Cell 94, 109–118. [37] Crease, D.J., Dyson, S. and Gurdon, J.B. (1998) Cooperation between the activin and Wnt pathways in the spatial control of organizer gene expression. Proc. Natl. Acad. Sci. USA 95, 4398– 4403. [38] Labbe´, E., Letamendia, A. and Attisano, L. (2000) Association of Smads with lymphoid enhancer binding factor 1/T cellspecific factor mediates cooperative signaling by the transforming growth factor- and Wnt pathways. Proc. Natl. Acad. Sci. USA 97, 8358. [39] Nishita, M., Hashimoto, M.K., Ogata, S. and Laurent, M.N. (2000) Interaction between Wnt and TGF-signalling pathways during formation of SpemannÕs organizer. Nature 403, 781–785. [40] Ishitani, T., Ninomiya-Tsuji, J., Nagai, S., Nishita, M., Meneghini, M., Barker, N., Waterman, M., Bowerman, B., Clevers, H., Shibuya, H. and Matsumoto, K. (1999) The TAK1-NLKMAPK-related pathway antagonizes signalling between b-catenin and transcription factor TCF. Nature 399, 798–802. [41] Furuhashi, M., Yagi, K., Yamamoto, H., Furukawa, Y., Shimada, S., Nakamura, Y., Kikuchi, A., Miyazono, K. and Kato, M. (2001) Axin facilitates Smad3 activation in the transforming growth factor signaling pathway. Cell. Biol. 21, 5132–5141. [42] Warner, D.R., Pisano, M.M., Roberts, E.A. and Greene, R.M. (2003) Identification of three novel Smad binding proteins involved in cell polarity. FEBS Lett. 539, 167–173. [43] Macias-Silva, M., Abdollah, S., Hoodless, P.A., Pirone, R., Attisano, L. and Wrana, J.L. (1996) MADR2 is a substrate of the TGFbeta receptor and its phosphorylation is required for nuclear accumulation and signaling. Cell 87, 1215–1224. [44] Shibamoto, S., Higano, K., Takada, R., Ito, F., Takeichi, M. and Takada, S. (1998) Cytoskeletal reorganization by soluble Wnt-3a protein signalling. Genes to Cells 3, 659–670. [45] Korinek, V., Barker, N., Morin, P.J., van Wichen, D., de Weger, R., Kinzler, K.W., Vogelstein, B. and Clevers, H. (1997) Constitutive Transcriptional Activation by a -Catenin-Tcf Complex in APC/ Colon Carcinoma. Science 275, 1784–1787. [46] Wrana, J.L., Attisano, L., Carcamo, J., Zentella, A., Doody, J., Laiho, M., Wang, X.F. and Massague´, J. (1992) TGF beta signals through a heteromeric protein kinase receptor complex. Cell 71, 1003–1014. [47] Stambolic, V., Ruel, L. and Woodgett, J.R. (1996) Lithium inhibits glycogen synthase kinase-3 activity and mimics Wingless signalling in intact cells. Curr. Biol. 6, 1664–1668. [48] Hu, M.C., Piscione, T.D. and Rosenblum, N.D. (2003) Elevated SMAD1/b-catenin molecular complexes and renal medullary cystic dysplasia in ALK3 transgenic mice. Development 130, 2753–2766. [49] G.J., H., D. Beach, p15INK4B is a potential effector of TGFbeta-induced cell cycle arrest. Nature 371 (1994) 257–261.

D.R. Warner et al. / FEBS Letters 579 (2005) 3539–3546 [50] Kuhnert, F., Davis, C.R., Wang, H.T., Chu, P., Lee, M., Yuan, J., Nusse, R. and Kuo, C.J. (2004) Essential requirement for Wnt signaling in proliferation of adult small intestine and colon revealed by adenoviral expression of Dickkopf-1. Proc. Natl. Acad. Sci. USA 101, 266–271. [51] Willert, K., Brown, J.D., Danenberg, E., Duncan, A.W., Weissman, I.L., Reya, T., Yates, J.R.r. and Nusse, R. (2003) Wnt proteins are lipid-modified and can act as stem cell growth factors. Nature 423, 448–452. [52] Linask, K.K., DÕAngelo, M., Gehris, A.L. and Greene, R.M. (1991) Transforming growth factor-beta receptor profiles of human and murine embryonic palate mesenchymal cells. Exp. Cell. Res. 192, 1–9. [53] Sun, G.Y., Navidi, M., Yoa, F.G., Lin, T.N., Orth, O.E., Stubbs, E.B.J. and MacQuarrie, R.A. (1992) Lithium effects on inositol phospholipids and inositol phosphates: evaluation of an in vivo model for assessing polyphosphoinositide turnover in brain. J. Neurochem. 58, 290–297. [54] Klein, T. and Arias, A.M. (1999) The vestigial gene product provides a molecular context for the interpretation of signals during the development of the wing in Drosophila. Development 126, 913–925. [55] Riese, J., Yu, X., Munnerlyn, A., Eresh, S., Hsu, S.C., Grosschedl, R. and Bienz, M. (1997) LEF-1, a nuclear factor coordinating signaling inputs from wingless and decapentaplegic. Cell 88, 777–787. [56] Labbe´, E., Letamendia, A. and Attisano, L. (2000) Association of Smads with lymphoid enhancer binding factor 1/T cell-specific factor mediates cooperative signaling by the transforming growth factor- and Wnt pathways. Proc. Natl. Acad. Sci. USA 97, 8358– 8363. [57] Tuli, R., Tuli, S., Nandi, S., Huang, X., Manner, P.A., Hozack, W.J., Danielson, K.G., Hall, D.J. and Tuan, R.S. (2003) Transforming growth factor-b-mediated chondrogenesis of human mesenchymal progenitor cells involves N-cadherin and mitogen-activated protein kinase and Wnt signaling cross-talk. J. Biol. Chem. 278, 41227–41236. [58] Waltzer, L. and Bienz, M. (1998) Drosophila CBP represses the transcription factor TCF to antagonize Wingless signalling. Nature 395, 521–525. [59] Hecht, A., Vleminckx, K., Stemmler, M.P., van Roy, F. and Kemler, R. (2000) The p300/CBP acetyltransferases function as transcriptional coactivators of beta-catenin in vertebrates. EMBO J. 19, 1839–1850. [60] Ito, Y., Yeo, J.Y., Chytil, A., Han, J., Bringas, P.J., Nakajima, A., Shuler, C.F., Moses, H.L. and Chai, Y. (2003) Conditional inactivation of Tgfbr2 in cranial neural crest causes cleft palate and calvaria defects. Development 130, 5269–5280. [61] Zhang, Z., Song, Y., Zhao, X., Zhang, X., Fermin, C. and Chen, Y. (2002) Rescue of cleft palate in Msx1-deficient mice by transgenic Bmp4 reveals a network of BMP and Shh signaling in the regulation of mammalian palatogenesis. Development 129, 4135–4146.