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Molecular Biology and Genetics |
Department of Cell and Developmental Biology, Weill Medical College of Cornell University, New York, New York 10021 [L. R. H., A. M. C. B.], and Strang Cancer Research Laboratory at the Rockefeller University, New York, New York 10021 [L. R. H., O. W., J. L., A. M. C. B.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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70% of mammary tumors from Wnt1 transgenic mice. A murine Twist promoter fragment was shown to be responsive to ß-catenin, and its activity was enhanced by coexpression of c-jun and Ets factors of the PEA3 family. Both PEA3 factors and c-jun were highly expressed in tumors from Wnt1 transgenic mice and may therefore contribute to the increased Twist expression observed in these tumors. To evaluate functional consequences of Twist induction, we examined the effect of Twist on mammary cell differentiation. Strikingly, overexpression of either Wnt1 or Twist in HC11 mammary epithelial cells completely suppressed induction of the milk protein ß-casein in response to lactogenic hormones. Additionally, Wnt1, but not Twist, partially abrogated induction of WDNM1, another marker of lactogenic differentiation. Taken together, our data indicate that Twist expression is regulated by Wnt/ß-catenin signaling and that both Wnt1 and Twist can function as inhibitors of lactogenic differentiation, an effect that could contribute to mammary tumorigenesis. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The canonical Wnt signaling pathway involves stabilization of a cytoplasmic ß-catenin pool (11
, 12)
, which can modulate transcription by interaction with transcription factors, including those of the TCF family (13
, 14)
. Inappropriate ß-catenin accumulation with consequent transcriptional activation can result from Wnt gene misexpression but also occurs as a consequence of mutation of the ß-catenin gene (CTNNB1) itself or of other components of the Wnt/ß-catenin signaling pathway such as AXIN or APC (10)
. Thus, there is considerable interest in identifying transcriptional targets of Wnt/ß-catenin signaling, particularly those that contribute to tumorigenesis. Several candidate genes have been identified, including cyclin D1 (15
, 16)
, c-myc (17)
, Matrilysin (18
, 19)
, PPAR
(20)
, WISP-1 (21)
, c-jun, and fra-1 (22)
. We are interested in identifying additional Wnt transcriptional targets and understanding their roles in Wnt-activated processes.
We focused on Twist as a candidate Wnt target gene based on evidence from Drosophila developmental genetics. Twist was first described in Drosophila as a gene essential for dorsoventral polarity (23 , 24) and encodes a transcription factor of the bHLH6 family. Ventral Twist expression in early embryos is regulated by the Rel factor Dorsal (25, 26, 27) . Additionally, Twist expression is diminished in Drosophila embryos that are deficient in wingless, the Drosophila homologue of Wnt1 (28) , suggesting that Twist might also be regulated by Wnt signaling. In mammals, Twist contributes to morphogenesis of the cranial neural tube: Twist-null mice die at E11.5 with unfused cranial neural folds, as well as defects of the head mesenchyme, somites, and limb buds (29) . Embryonic Twist expression patterns are consistent with contributions to neural and limb development (30, 31, 32) . Germ-line mutations at the TWIST locus in humans cause Saethre-Chotzen syndrome, an autosomal-dominant craniosynostosis syndrome that results in premature closure of the coronal sutures of the skull (33 , 34) . Additionally, Twist shows some oncogenic properties, promoting colony formation of mouse embryonic fibroblasts in soft agar and antagonizing p53-induced growth arrest (35) .
Here we demonstrate that Twist is up-regulated in response to Wnt1 expression in mouse mammary epithelial cell lines and tumors. Analysis of Twist promoter regulation revealed responsiveness to ß-catenin, c-jun, and Ets factors of the PEA3 family. All of these are elevated in Wnt1-expressing mammary tumors and may therefore contribute to the observed Twist up-regulation in these tumors. We also found that overexpression of either Wnt1 or Twist abrogated prolactin-stimulated ß-casein induction in a cell culture model. Furthermore, up-regulation of an additional lactogenic marker, WDNM1, was also diminished in Wnt1-overexpressing cells. Since suppression of terminal differentiation is associated with tumorigenesis, Wnt-mediated suppression of lactogenic differentiation could contribute to Wnt-induced mammary tumorigenesis.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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9 kb was isolated and subcloned into pBluescript SK+ (Stratagene). A 2-kb NgoMI fragment containing 70 bases from exon 1 (30)
plus 1.9 kb of upstream sequence was subcloned from this construct into SmaI-cut pGL2basic (Promega, Madison, WI) to generate the promoter reporter construct Twist-LUC. An additional promoter reporter construct Rev-Twist-LUC was also generated in which the 2-kb promoter fragment was inserted in the reverse orientation. pMV-Twist was constructed by subcloning a PCR product encompassing the entire coding region of Twist into the retroviral vector pMV7 (PCR primers; 5'-CCGGAATTCATGATGCAGGACGT-3' and 5'-CCGGAATTCCTAGTGGGACGCGGA-3'). pSK-Twist was generated by subcloning a blunted 859-bp SacII fragment of genomic Twist comprising the entire Twist open reading frame into NotI-digested pBluescript SK+ using NotI linkers. pRcTwist was constructed by subcloning a BamHI-AvrII fragment from pSK-Twist containing the entire coding sequence of Twist into pRcCMV (Invitrogen, Carlsbad, CA). All constructs were verified by DNA sequencing by the DNA/Protein Technology Center (Rockefeller University, New York, NY). The plasmids used for transfection of 293 cells (pMT23, pMT23ß-catenin, pCAN
N89ß-catenin, pCANmycPEA3, pcDNA-ER81, pCANmycERM, Ets-1, pSG5-Ets-2, pCMX-c-jun, pRL-TK, and the stromelysin-1 promoter construct p754TR-Luc) were as described previously (18
, 36)
.
Mouse Tissue and Tumor Harvesting.
A breeding colony of Wnt1 transgenic mice (4)
was maintained by crossing Wnt1 transgenic B6/SJL males (Jackson Laboratory) with strain-matched females. Mice were genotyped by PCR analysis of tail-tip DNA, as described previously (36)
. Wnt1 transgenic animals were sacrificed when tumors were 1 cm in diameter, and wild-type littermates were simultaneously sacrificed. Tumors and MGs were snap-frozen in liquid nitrogen and stored at -80°C before RNA preparation.
RNA Preparation and Northern Blotting.
RNA was prepared from tissue or cells using TRIzol Reagent (Life Technologies, Inc., Grand Island, NY) or RNAzol B (Tel-Test, Inc., Friendswood, TX) according to the manufacturers’ instructions. Northern blots and hybridization were performed as previously described (37)
using 20 µg of total RNA. The Twist probe was a 421-bp XbaI-EcoRI fragment from the 3'-untranslated region of mouse Twist cDNA (30)
. The murine ß-casein probe was prepared by PCR amplification of a 306-bp fragment (bases 437–742 of the mouse genomic DNA), which was subcloned into pBluescript (Stratagene). Murine GAPDH probe was obtained from Alan Ashworth (Institute of Cancer Research, London, United Kingdom). GAPDH was used to control for loading of each lane. c-jun probe (hCJ-1) was obtained from Andrew S. Kraft (University of Colorado Health Sciences Center, Denver, CO). Murine WDNM1 and WAP probes (pBS-WDNM1 and pBS-WAP) were obtained from Gertraud Robinson (National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD). Autoradiographic exposures of Northern blots were quantitated by analysis on a Macintosh computer using the public domain NIH Image program (developed at the United States NIH and available online).7
Values obtained were normalized to those obtained for GAPDH.
Cell Culture and Transfection.
Generation of C57/MV7 and C57/Wnt-1 cell lines, and culture conditions of these and C57MG cells, have been described previously (37
, 38)
. Culture and transfection of 293 human embryonic kidney cells was performed as described previously (36)
. Lysates were prepared 48 h after transfection, and Firefly and Renilla luciferase activities were measured using a Dual Luciferase Reagent kit (Promega) and a Monolight 2010 luminometer (Analytical Luminescence Laboratory). HC11 mouse mammary epithelial cells (39)
were grown in RPMI 1640 containing 8% FBS, 10 ng/ml murine epidermal growth factor, 5 µg/ml bovine insulin, 100 units/ml penicillin, and 100 µg/ml streptomycin. Cells were infected with MV7, MVWnt1, and MVTwist retrovirus using helper-free virus stocks as described previously (40)
. Approximately 200 G418-resistant colonies were pooled to generate the pooled populations designated HC11/MV7, HC11/Wnt-1, and HC11/Twist. Lactogenic stimulation of HC11-derived cell lines was achieved by maintaining the cells at confluence for 2 days, then incubating for an additional 3 days in RPMI 1640 containing 8% FBS, 1 µM dexamethasone, 5 µg/ml insulin, and 5 µg/ml bovine prolactin. Cos cells were grown in DMEM containing 10% FBS, 100 units/ml penicillin, and 100 µg/ml streptomycin and were transfected with Lipofectamine (Life Technologies, Inc.) according to the manufacturer’s instructions.
Generation of Antisera.
A peptide corresponding to amino acids 34–46 of Twist was synthesized with an additional COOH-terminal cysteine residue (Peptide Core Facility; Rockefeller University) and conjugated to Keyhole Limpet Hemocyanin using a coupling kit (Pierce, Rockford, IL). Three rabbits were immunized with peptide conjugate and sera collected (Covance Research Products, Philadelphia, PA). Sera were tested for their ability to recognize Twist protein on a Western blot using bacterially produced recombinant protein (glutathione S-transferase versus glutathione S-transferase-Twist) and Twist protein ectopically expressed in Cos cells. Serum from no. 535 was selected for additional use.
Cell Lysate Preparation and Analysis.
Crude cell lysates were prepared by removing the cells from the dishes, pelletting, and resuspending in lysis buffer containing 150 mM NaCl, 100 mM Tris (pH 7.4), 1% Tween 20, 1 mM EDTA, 20% glycerol, and Complete Proteinase Inhibitor (Boehringer Mannheim, Indianapolis, IN). Resuspended cell pellets were vortexed briefly and incubated 30 min, 4°C, then sonicated for a total of 30 s. Debris was pelletted by brief centrifugation, and the supernatant was stored at -20°C.
For Western analysis, samples were subjected to SDS-PAGE using 4–15% gradient gels (Bio-Rad, Hercules, CA) and proteins transferred to polyvinylidene fluoride membrane (Immobilon, Millipore, Billerica, MA). Blots were blocked with 3% gelatin in Tris-buffered saline [150 mM NaCl, 10 mM Tris, (pH 8.0)] and incubated 1 h in primary antibody diluted 1:1000 or 1:3000 in 1% gelatin in Tris-buffered saline. Secondary antibody was alkaline phosphatase-conjugated goat antirabbit antibody (Bio-Rad). Blots were developed using a chemiluminescent reagent (NEN, Boston, MA).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, and a corresponding increase in Twist protein was observed (Fig. 1B)
. We had previously made the novel observation that Twist is expressed in adult murine MG during a PCR-based screen designed to identify bHLH proteins involved in the process of mammary differentiation (data not shown). Therefore, we also examined Twist expression in wild-type MG and in mammary tumors from Wnt1 transgenic mice. Interestingly, we observed increased expression of Twist RNA in five of seven tumors tested relative to that observed in normal MG (Fig. 1, C and D)
. Taken together, these data suggest that Twist is regulated by Wnt signaling.
|
. At the 5' end, our sequence extends the previously characterized murine sequence (30)
by 1280 bases. At the 3' end, the fragment extends into exon 1 but contains no coding sequence.
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200% in response to ß-catenin overexpression (Fig. 3, A and B
; P < 0.001). In contrast, the construct Rev-Twist-LUC in which the Twist promoter fragment is inserted in the reverse orientation was unresponsive to ß-catenin (Fig. 3A)
, demonstrating specificity of the Twist-LUC response.
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. However, both c-jun and ß-catenin synergistically increased Twist-LUC activity when coexpressed with PEA3 (P < 0.01). Strikingly, coexpression of all three transcription factors increased Twist promoter activity to 840% of control (P < 0.001). We also examined the response of the Twist promoter to the Ets factors ETS-1 and ETS-2 in comparison with the PEA3 subfamily members PEA3, ER81, and ERM. Of these, PEA3 and ERM were the most potent activators of the Twist promoter (Fig. 4)
. ER81 and ETS-1 elicited weaker responses, and ETS-2 caused no activation. It was not possible to directly compare expression levels of the various factors because the cDNAs were not uniformly epitope-tagged. However, because ETS-2 caused much stronger activation of the stromelysin-1 promoter than did PEA3 in the same experiment (Fig. 4
, right panel), these data suggest that the Twist promoter is preferentially responsive to PEA3 subfamily members.
|
. To test the involvement of c-jun, we analyzed c-jun expression in Wnt1-expressing C57MG cells and mammary tumors. Although c-jun transcription can be activated by ß-catenin in human colorectal carcinoma cell lines (22)
, we did not observe increased c-jun expression in C57/Wnt-1 relative to control C57/MV7 cells (data not shown). However, c-jun was highly expressed in all six tumors tested (Fig. 5)
. These data indicate that both c-jun and PEA3 factors may contribute to the Twist up-regulation observed in a subset of Wnt1 transgenic mammary tumors. However, because both c-jun and PEA3 are uniformly expressed at high levels in all tumors tested (Fig. 5
; Ref. 36
), whereas Twist expression was increased in only five of seven tumors examined (Fig. 1, C and D)
, the murine Twist promoter may also be subject to regulation by other factors yet to be identified.
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. No Twist protein was detectable in the control infected HC11/MV7 cells or in the parental HC11 cells (Fig. 6A)
.
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. DIP-mediated up-regulation of the WDNM1 transcript was unaffected by Twist overexpression (Fig. 6B)
. Strikingly, however, induction of ß-casein RNA in response to DIP was completely absent in HC11/Twist cells (Fig. 6C)
. Thus, overexpression of Twist was sufficient to block hormone-stimulated ß-casein expression in HC11 cells.
Ectopic Wnt1 Expression Antagonizes ß-Casein Induction.
Taken together, our observations that Twist can regulate ß-casein induction and that Twist itself is regulated by Wnt signaling suggested that Wnt1 might also modulate cellular responsiveness to lactogenic hormones. To test this possibility, we generated Wnt1-expressing HC11 cells (HC11/Wnt-1) and control cells (HC11/MV7) using retroviral expression vectors. Interestingly, Wnt1 expression in HC11 cells led to morphological transformation (data not shown), as has previously been described for both C57MG and RAC311 cells (5
, 6)
. Expression of Wnt1 in HC11 cells caused an increase in Twist protein and RNA (Fig. 7, A and B)
, consistent with our observations in C57MG-derived cell lines (Fig. 1)
. The effect of Wnt1 expression on both ß-casein and WDNM1 induction was compared in HC11/MV7 and HC11/Wnt-1 cells, again using DIP as a lactogenic stimulus. Control HC11/MV7 cells showed robust WDNM1 expression in response to DIP, but Wnt1 overexpression partially abrogated WDNM1 induction in response to hormonal stimulation (Fig. 7C)
. Additionally, we examined the effect of Wnt1 overexpression on ß-casein induction (Fig. 7D)
. Although ß-casein was up-regulated by DIP in control HC11/MV7 cells, there was no induction of ß-casein in HC11/Wnt-1 cells. Thus, Wnt1 expression, similarly to Twist overexpression, completely abolished the ability of HC11 cells to activate ß-casein transcription in response to DIP. Because Wnt1 causes induction of Twist in these cells, their failure to respond to lactogenic stimuli by activating ß-casein transcription may thus be attributable to Twist.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and 7)
. Additionally, we have shown that Twist is expressed in adult murine MG and that Twist expression is significantly increased in mammary tumors from Wnt1 transgenic mice (Fig. 1)
. To our knowledge, this is the first demonstration that Twist is up-regulated in response to Wnt signaling and that Twist is expressed in mammary tissue. Several Wnt genes exhibit dynamic expression patterns during mammary development (47, 48, 49, 50)
. In particular, the mammary expression profile of Wnt2 resembles that which we have observed for Twist (48)
.8
Wnt2 activates the canonical Wnt/ß-catenin signaling pathway (12
, 51)
, and we have observed elevated Twist expression in C57/Wnt2 cells (data not shown). Taken together, these data suggest that Wnt2 could contribute to the regulation of endogenous Twist expression in the MG.
Having demonstrated Wnt1-mediated Twist up-regulation, we next investigated the mechanism by which Wnt1 transcriptionally activates Twist. A Twist promoter reporter construct was generated containing 2.0 kb of 5' untranslated sequence from the murine Twist gene (Fig. 2)
linked to a luciferase reporter gene. This promoter fragment contains three potential TCF binding sites (Fig. 2B)
. In addition, we identified 34 potential Ets binding sites based on the presence of the core binding site 5'-GGA,A/T-3', of which approximately one-third were conserved between mouse and human. Analysis of the sequence using MatInspector9
revealed several AP1 sites and numerous other potential transcription factor binding sites. Luciferase assays demonstrated that this Twist promoter construct was responsive to ß-catenin (Fig. 3)
. Additionally, Ets factors of the PEA3 subfamily activated the Twist promoter, and c-jun enhanced the responses to both ß-catenin and PEA3 (Figs. 3
and 4)
. The synergy between ß-catenin, PEA3, and c-jun is particularly striking because similar findings have recently been reported with respect to the Matrilysin promoter, an additional target of Wnt/ß-catenin signaling (18
, 43)
. We have also shown that PEA3 factors activate transcription of the cyclooxygenase 2 gene (36)
, another gene that is responsive to Wnt signaling (37
, 41)
. Taken together, these findings suggest that PEA3 factors may contribute to regulation of multiple target genes of the Wnt/ß-catenin pathway. Consistent with this hypothesis, the promoters of several genes known to be ß-catenin-responsive also contain consensus Ets binding sites. For example, the Drosophila gene Even-skipped is coordinately regulated via TCF and Ets binding sites (52)
, and the cyclin D1 promoter can be activated cooperatively by PEA3, ß-catenin, and c-jun10
. Interestingly, the promoter of the Drosophila Twist gene also contains both Ets and TCF binding sites,11
suggesting that there may be a role for ß-catenin and Ets factors in regulating its expression. This would represent a novel mechanism of Drosophila Twist activation, in addition to the previously established Dorsal-mediated regulation (25, 26, 27)
.
Both Wnt1 and Twist Inhibit Mammary Cell Differentiation.
Twist and Wnt1 have previously been shown to antagonize the differentiation of certain cell lineages (44
, 45
, 53
, 54)
. Therefore, we were interested in determining whether either Twist or Wnt1 might repress lactogenic differentiation in mammary cells. To address this possibility, responsiveness to lactogenic hormones was examined in HC11 cells, using the milk protein transcripts ß-casein and WDNM1 as markers of lactogenic differentiation. Overexpression of Wnt1, but not of Twist, diminished WDNM1 up-regulation in response to lactogenic stimuli (Figs. 6
and 7)
. In contrast, ß-casein induction was effectively abolished by either Wnt1 or Twist (Figs. 6
and 7)
. Interestingly, the expression profile of Twist during postnatal murine mammary development is consistent with a role for Twist in suppressing ß-casein expression in vivo. Mammary Twist expression is constant during early gestation but diminishes during mid pregnancy, exhibiting a reciprocal expression pattern with the milk protein ß-casein.8
Taken together, these observations raise the possibility that Twist may function in vivo to negatively regulate ß-casein expression.
The simplest interpretation of the ability of Twist to antagonize ß-casein induction is that Twist is acting as a transcriptional repressor. Several mechanisms have been proposed to account for transcriptional repression mediated by mammalian Twist, mostly through analysis of its role in myogenic differentiation (55, 56, 57) . Additionally, the ability of Twist to bind to and inhibit histone acetyl transferases may be important for Twist-mediated transcriptional repression (58) .
Suppression of Differentiation and Tumorigenesis.
The ability of Wnt1 to inhibit differentiation, as evidenced by diminished induction of lactogenic markers, may be a contributory factor to Wnt-mediated tumorigenesis. Suppression of terminal differentiation is thought to favor tumorigenesis, a paradigm particularly well illustrated by the HLH-containing Id proteins. Originally identified as inhibitors of myogenic differentiation, Id proteins are now known to function as dominant negative regulators of cell lineage commitment and differentiation in multiple cell types (59
, 60)
. However, Id proteins are also associated with tumorigenesis (61, 62, 63)
, and indeed, expression of an Id1 transgene can induce formation of intestinal adenomas (64)
. By analogy, the ability of Wnt1 to suppress lactogenic differentiation may be important for its function as a mammary oncogene. This effect of Wnt1 may be mediated in part via Twist up-regulation because overexpressed Twist completely abolishes ß-casein induction. However the failure of Twist to repress WDNM1 induction clearly suggests a requirement for other factors to mediate this effect. In addition to altered differentiation, enhanced cellular proliferation and diminished apoptosis are frequently prerequisites for carcinogenesis. Of the Wnt/ß-catenin transcriptional targets thus far identified, several are likely to contribute to these processes (15, 16, 17
, 20, 21, 22)
. Thus, suppression of terminal differentiation by Wnt1 is only one of a panoply of responses that may contribute to Wnt-mediated tumorigenesis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by NIH Grant CA47207 (to A. M. C. B.) and by donations to Strang Cancer Prevention Center from the Fashion Footwear Association of New York. 
2 To whom requests for reprints should be addressed, at Strang Cancer Research Laboratory, 1230 York Avenue, Box 231, New York, NY 10021. E-mail: lrhowe{at}med.cornell.edu
3 These authors contributed equally to this work.
4 Present address: Tokyo Women’s Medical University Daini Hospital, 2-1-10 Nishiogu, Arakawa-ku, Tokyo 116-8567, Japan.
5 Present address: Arena Pharmaceuticals, San Diego, CA 92121.
6 The abbreviations used are: bHLH, basic helix-loop-helix; MG, mammary gland; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DIP, dexamethasone/insulin/prolactin; FBS, fetal bovine serum; WAP, whey acidic protein.
7 Internet address: rsb.info.nih.gov/nih-image.
8 O. Watanabe and J. Leonard, unpublished data.
9 Internet address: transfac.gbf.de/.
10 C. Messier and J. A. Hassell, personal communication.
11 V. Cox and M. K. Baylies, personal communication.
Received 4/18/02. Accepted 2/14/03.
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 Y.-Q. Zhu and X.-D. Tan TFF3 modulates NF-{kappa}B and a novel negative regulatory molecule of NF-{kappa}B in intestinal epithelial cells via a mechanism distinct from TNF-{alpha} Am J Physiol Cell Physiol, November 1, 2005; 289(5): C1085 - C1093. [Abstract] [Full Text] [PDF]
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Y. Yin, R. G. Russell, L. E. Dettin, R. Bai, Z.-L. Wei, A. P. Kozikowski, L. Kopleovich, and R. I. Glazer Peroxisome Proliferator-Activated Receptor {delta} and {gamma} Agonists Differentially Alter Tumor Differentiation and Progression during Mammary Carcinogenesis Cancer Res., May 1, 2005; 65(9): 3950 - 3957. [Abstract] [Full Text] [PDF]
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 T. Sakatani, A. Kaneda, C. A. Iacobuzio-Donahue, M. G. Carter, S. de Boom Witzel, H. Okano, M. S. H. Ko, R. Ohlsson, D. L. Longo, and A. P. Feinberg Loss of Imprinting of Igf2 Alters Intestinal Maturation and Tumorigenesis in Mice Science, March 25, 2005; 307(5717): 1976 - 1978. [Abstract] [Full Text] [PDF]
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 C. N. Bennett, K. A. Longo, W. S. Wright, L. J. Suva, T. F. Lane, K. D. Hankenson, and O. A. MacDougald Regulation of osteoblastogenesis and bone mass by Wnt10b PNAS, March 1, 2005; 102(9): 3324 - 3329. [Abstract] [Full Text] [PDF]
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M. El-Tanani, A. Platt-Higgins, P. S. Rudland, and F. C. Campbell Ets Gene PEA3 Cooperates with {beta}-Catenin-Lef-1 and c-Jun in Regulation of Osteopontin Transcription J. Biol. Chem., May 14, 2004; 279(20): 20794 - 20806. [Abstract] [Full Text] [PDF]
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), exons (
), and potential TATA boxes (black rectangles), of which only the 3' TATA box is functional in human Twist 
) or the construct Rev-Twist-LUC (
) in which the Twist promoter fragment is inserted in the reverse orientation. pRL-TK was cotransfected as an internal control. Results shown are the mean + SD of 6 replicates from a representative experiment expressed relative to activity in control cells transfected with empty expression vectors. B, 293 cells were transfected with combinations of expression vectors encoding c-jun, ß-catenin (ß-Cat), and PEA3 plus Twist-LUC and pRL-TK, and luciferase assays were performed as described above. Results shown are the mean + SD of 6 replicates.
