Regulation of Cyclooxygenase-2 and Periostin by Wnt-3 in Mouse Mammary Epithelial Cells

Wnt family members are critical in developmental processes and have been shown to promote carcinogenesis when ectopically expressed in the mouse mammary gland. The gene expression pattern mediated by Wnt is pivotal for these diverse responses. The Wnt pathway has been conserved among different species. Genetic studies have shown that Wnt effects are mediated, at least in part, by β-catenin, which regulates transcription of “downstream genes.” Wnt stimulation inactivates glycogen-synthase kinase-3β (GSK-3) with subsequent stabilization of β-catenin, which after heterodimerizing with lymphocyte enhancer factor-1/T-cell factor cofactors stimulates transcription. To establish whether Wnt-stimulated transcription is mediated solely by β-catenin, a comparison was made of gene expression profiles in response to Wnt-3, overexpression of β-catenin, and inhibition of GSK-3. Infection of cells with Wnt-3 and inhibition of GSK-3 regulate a set of genes that include cyclooxygenase-2 and periostin. Interestingly, overexpression of β-catenin or reducing β-catenin levels with antisense oligonucleotide transfection did not have any effect on cyclooxygenase-2 or periostin expression, thereby defining a Wnt pathway, which cannot be mimicked by β-catenin overexpression. Wnt family members are critical in developmental processes and have been shown to promote carcinogenesis when ectopically expressed in the mouse mammary gland. The gene expression pattern mediated by Wnt is pivotal for these diverse responses. The Wnt pathway has been conserved among different species. Genetic studies have shown that Wnt effects are mediated, at least in part, by β-catenin, which regulates transcription of “downstream genes.” Wnt stimulation inactivates glycogen-synthase kinase-3β (GSK-3) with subsequent stabilization of β-catenin, which after heterodimerizing with lymphocyte enhancer factor-1/T-cell factor cofactors stimulates transcription. To establish whether Wnt-stimulated transcription is mediated solely by β-catenin, a comparison was made of gene expression profiles in response to Wnt-3, overexpression of β-catenin, and inhibition of GSK-3. Infection of cells with Wnt-3 and inhibition of GSK-3 regulate a set of genes that include cyclooxygenase-2 and periostin. Interestingly, overexpression of β-catenin or reducing β-catenin levels with antisense oligonucleotide transfection did not have any effect on cyclooxygenase-2 or periostin expression, thereby defining a Wnt pathway, which cannot be mimicked by β-catenin overexpression. adenomatous polyposis coli glycogen-synthase kinase polymerase chain reaction reverse transcriptase 4-morpholineethanesulfonic acid prostaglandin E2 cyclooxygenase glyceraldehyde-3-phosphate dehydrogenase lymphocyte enhancer factor-1 The Wnt proteins are a family of secreted cysteine-rich glycoproteins that play an essential role in directing developmental processes such as cell adhesion, cell fate, and cell proliferation (1Nusse R. Varmus H.E. Cell. 1992; 69: 1073-1087Abstract Full Text PDF PubMed Scopus (830) Google Scholar,2Moon R.T. Brown J.D. Torres M. Trends Genet. 1997; 13: 157-162Abstract Full Text PDF PubMed Scopus (546) Google Scholar). The Wnt signaling pathway appears to be highly conserved across different species, and genetic and biochemical studies inCaenorhabditis elegans, Drosophila,Xenopus and mammals have contributed to increased understanding of the pathway (for review see Ref. 3Cadigan K.M. Nusse R. Genes Dev. 1997; 11: 3286-3305Crossref PubMed Scopus (2225) Google Scholar). Some of the Wnt genes have been shown to promote mammalian carcinogenesis. Wnt-1 and Wnt-3 were initially identified as mouse mammary oncogenes that became tumorigenic by the insertion of a mouse mammary tumor virus (4Nusse R. Varmus H.E. Cell. 1982; 31: 99-109Abstract Full Text PDF PubMed Scopus (1251) Google Scholar, 5Roelink H. Wagenaar E. Lopes da Silva S. Nusse R. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4519-4523Crossref PubMed Scopus (158) Google Scholar). Even though a role for Wnt proteins in breast tumorigenesis has been established in mice, this link has not yet been made in human breast cancer. However, components of the Wnt signaling pathway such as adenomatous polyposis coli (APC),1 a tumor suppressor protein, and β-catenin are clearly involved in other forms of human cancers including melanoma, colon, and hepatocellular cancer (6Polakis P. Curr. Opin. Genet. Dev. 1999; 9: 15-21Crossref PubMed Scopus (605) Google Scholar). Stimulation of cells with Wnt-1 protein results in an increase of cytosolic levels of β-catenin as a consequence of the inhibition of glycogen-synthase kinase-3β. β-Catenin then heterodimerizes with a member of the Lef-1/T-cell factor family of transcription factors and induces gene transcription. APC can bind to β-catenin and facilitate its degradation when phosphorylated by GSK-3. Deletions in the tumor suppressor protein APC found in colon cancer preclude β-catenin degradation, and in so doing the increased level of β-catenin can stimulate transcription as described above. Further, stabilizing mutations or deletions in the regulatory N-terminal domain of β-catenin also result in increased transcription (3Cadigan K.M. Nusse R. Genes Dev. 1997; 11: 3286-3305Crossref PubMed Scopus (2225) Google Scholar). How this increase in transcription relates to cancer is unclear, but some recently identified β-catenin target genes cyclin D1 and the protooncogene c-myc may contribute to neoplastic transformation (7Tetsu O. McCormick F. Nature. 1999; 398: 422-426Crossref PubMed Scopus (3253) Google Scholar, 8He T.C. Sparks A.B. Rago C. Hermeking H. Zawel L. da Costa L.T. Morin P.J. Vogelstein B. Kinzler K.W. Science. 1998; 281: 1509-1512Crossref PubMed Scopus (4075) Google Scholar). A number of potential Wnt-1 target genes have been identified in different organisms, including ultrabithorax andengrailed in Drosophila (9Riese J., Yu, X. Munnerlyn A. Eresh S. Hsu S.C. Grosschedl R. Bienz M. Cell. 1997; 88: 777-787Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar, 10Hooper J.E. Nature. 1994; 372: 461-464Crossref PubMed Scopus (111) Google Scholar), nodal-related 3and siamois in Xenopus (11McKendry R. Hsu S.C. Harland R.M. Grosschedl R. Dev. Biol. 1997; 192: 420-431Crossref PubMed Scopus (212) Google Scholar, 12Brannon M. Kimelman D. Dev. Biol. 1996; 180: 344-347Crossref PubMed Scopus (122) Google Scholar, 13Carnac G. Kodjabachian L. Gurdon J.B. Lemaire P. Development. 1996; 122: 3055-3065PubMed Google Scholar),Connexin 43 (14), Wisp (15Pennica D. Swanson T.A. Welsh J.W. Roy M.A. Lawrence D.A. Lee J. Brush J. Taneyhill L.A. Deuel B. Lew M. Watanabe C. Cohen R.L. Melhem M.F. Finley G.G. Quirke P. Goddard A.D. Hillan K.J. Gurney A.L. Botstein D. Levine A.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14717-14722Crossref PubMed Scopus (459) Google Scholar), cyclin D1(16Issack P.S. Ziff E.B. Cell Growth Differ. 1998; 9: 837-845PubMed Google Scholar), and Cox-2 (17Howe L.R. Subbaramaiah K. Chung W.J. Dannenberg A.J. Brown A.M. Cancer Res. 1999; 59: 1572-1577PubMed Google Scholar) in various mammalian cell lines or tissues. Furthermore, it is not clear whether the entire transcriptional response to the stimulation by Wnt-1 or Wnt-3 is mediated by β-catenin-dependent processes. To explore whether there is any evidence for Wnt-stimulated pathways that are independent of β-catenin, we compared gene profiles regulated by Wnt-3, β-catenin, and inhibition of GSK-3. Cox-2 was up-regulated and periostin (formerly named osteoblast-specific factor-2) was down-regulated by both Wnt-3 and inhibition of GSK-3. However, in contrast, β-catenin did not have an effect on the regulation of these two genes, even though it did affect expression of other genes. Importantly, the data show that the genes regulated by Wnt-3 involve multiple pathways downstream of GSK-3, not all of which are dependent on β-catenin. The mouse mammary epithelial cell line C57MG was grown in Dulbecco's modified Eagle's medium (4.5 mg/mld-glucose) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, 2 mm l-glutamine, and 10 μg/ml insulin (Sigma). Wnt-3 was expressed in C57MG cells after infection with a murine leukemia virus-based retroviral vector (LNCX, Invitrogen). The vector expresses Wnt-3, which was cloned from a mouse brain library, under the control of the cytomegalovirus promoter and the bacterial neomycin phoshotransferase gene (neo). The same vector was used for expression of a β-catenin deletion mutant (ΔN89β-catenin) that lacks the potentially destabilizing phosphorylation sites for GSK-3. Infected cell populations were selected for 3 weeks in 400 μg/ml Geneticin (G418). At that time partial transformation to a more spindle-like cell shape was easily detectable in the Wnt-3 cells. About 400 G418 resistant clones designated Wnt-3, ΔN89β-catenin, or control (empty vector) were pooled and expanded for total RNA preparation and protein lysate preparation. Total RNA was isolated from Wnt-3 or vector control cells according to manufacturer's conditions (Qiagen) and then further treated with DNase using the MessageClean (Genhunter) protocol. Based on a method developed by Liang and Pardee (18Liang P. Pardee A.B. Science. 1992; 257: 967-971Crossref PubMed Scopus (4694) Google Scholar), 20 primers provided by the Hieroglyph mRNA profile kit for differential display analysis (Genomyx Corp.) were used to amplify 12 different pools of reverse-transcribed PCR reactions. A total of 240 primer pairs were used to profile gene expression. Products from these PCR reactions were run in duplicate with two sets of independently prepared total RNA on 4.5% sequencing gels using a genomyx LR sequencer (Genomyx Corp.). The dried gels were exposed to Kodak XAR-2 film (Eastman Kodak Co.). A total of 50 differentially expressed cDNA fragments were excised, reamplified according to the manufacturer's protocol (Genomyx Corp.), and sequenced. The length of the PCR products ranged from 0.3 to 1.0 kilobases. Only bands that were reproduced in duplicate lanes for two independent total RNA preparations were excised and reamplified. The cDNA microarray consisted of 26 cDNA fragments identified in the differential display analysis and control genes such as actin and tubulin. Poly(A)+ RNA was purified with the Oligotex mRNA mini kit (Qiagen) from total RNA isolated from Wnt-3-expressing and control cells. 0.5 μg of mRNA was oligo(dT)- and nonamer-primed at 70 °C for 5 min, cooled on ice, and then reverse-transcribed with 600 units of SuperscriptII reverse transcriptase for 2 h at 42 °C in 1× first strand buffer, 10 mm dithiothreitol, 2 mmeach dNTP, 1 mm dCTP (all reagents, Life Technologies, Inc.), and either 1 mm Cy3 or Cy5 (Amersham Pharmacia Biotech) for fluorescent labeling of the cDNA in a 20-μl total volume. The mRNA was digested with 1 unit RNaseH (Life Technologies Inc.) and 0.5 unit RNase ONE (Promega) for 30 min at 37 °C. Unincorporated nucleotides were removed using Qiaquick PCR clean up spin columns (Qiagen), and the volume was reduced to 9.5 μl. The aminosilane-coated slides were prehybridized for 3–4 h at 42 °C in 5× SSC, 0.1% SDS, and 50% formamide solution after UV-cross-linking and baking of the slides for 1 h at 80 °C. The probe was made in 5× SSC, 0.1% SDS, 50% formamide, 1 μg COT DNA (Roche Molecular Biochemicals), 1.25 μg of poly(A) oligo, and 4.25 μl of the fluorescently labeled cDNA in a volume of 25 μl and hybridized to the slides overnight at 42 °C. The next day the slides were washed once in 1× SSC, 0.2% SDS, twice in 2× 0.1 SSC, 0.2% SDS, and dried after a wash in deionized water. Fluorescence intensities of the immobilized probes were determined from images taken with an Arrayscanner GenerationII (Molecular Dynamics) confocal microscope with laser excitation sources and interference filters appropriate for Cy3 and Cy5 fluors. Separate scans were taken for each fluor at the resolution of 80 μm2/pixel. The image intensities were quantified using ImageQuant software (Molecular Dynamics). A minimal threshold value was set, and spots with values below this threshold were considered negative. The average for the 16 repeats of immobilized probes was taken to calculate the average intensities. The of the average intensities determined the of gene expression in the different and actin to cells were treated with of for were prepared by cells with buffer, of mm and 1 mm The were run (Qiagen) and at °C cytosolic cells were in mm mm mm 10 mm and (Roche Molecular by at for 10 min, were in a at for min at °C. The which the cytosolic was and were prepared for was under reducing conditions on gels or using gels with proteins were from the (Amersham Pharmacia were with β-catenin mouse Cox-2 and The were then with a by with an (Amersham Pharmacia Total RNA was isolated from C57MG cells using the kit μg of each RNA was by to and by to Cox-2 and probes to nucleotides and were by PCR and labeled with according to the manufacturer's protocol DNA The and was by the protocol for the and kit which a for of mRNA to in RNA for each cells were grown overnight in The cells were with and using (Life Technologies, h and after cell (Promega) was To GSK-3 kinase was in 50 mm 1 mm dithiothreitol, and 10 mm with μg of protein with of The kinase reaction was by an including a at the reaction was The filters were washed in and and was in a kinase reaction was in mouse β-catenin or reverse control control were C57MG cells at a of 100 by using a J.E. J.D. Cohen Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus Google Scholar). were for in RNA buffer, protein for or for the gene a reaction kit 1 μg of total RNA from different antisense oligonucleotide transfection was reverse 10% of the reaction was in a PCR using the the was quantified using a that each number at which the of the was in the with a value was then to the value of the or actin for each cells were grown for h in medium was for 5 min at μl of the was for prostaglandin E2 in an according to the manufacturer's protocol (Amersham Pharmacia To Wnt-3 target genes, a reverse differential display analysis was used to mRNA levels from two C57MG cell lines that were with either Wnt-3 or control The mammary epithelial cell line C57MG a response to expression of Wnt-3 and other Wnt proteins H. M.A. M. Brown A.M. J. Cell Growth Differ. 1997; Google Scholar) and has been used for studies on the Wnt The cells an spindle-like cell shape and show increased levels of cytosolic β-catenin 1 in response to Wnt-3 expression. of total RNA from Wnt-3 cells and from control cells were reverse-transcribed and in PCR reactions with 240 primer pairs to profile gene expression. of the 26 that were differentially were up-regulated and were down-regulated in Wnt-3 cells control cells. 26 genes, were were and was fragments were considered as a with any gene in the data The differential expression pattern was by microarray and the microarray the 26 cDNA fragments were to a labeled cDNA probes from Wnt-3 and control Cox-2 a up-regulated and periostin a down-regulated in data were by analysis of mRNA from Wnt-3 and C57MG cells and periostin mRNA levels did not in response to overexpression 3 analysis regulation of Cox-2 and periostin in Wnt-3 cells. Total RNA was prepared from and μg of each RNA was by as described under and The was the was with Cox-2 or murine periostin and and the with a murine periostin mRNA level is in image the levels of mRNA were in protein levels was for Cox-2 in prepared from Wnt-3-expressing cells when compared with from vector control cells of periostin protein not be were C57MG cells ectopically did not have increased Cox-2 protein levels The of in the was by analysis and its transcriptional was as a increase in the gene expression medium from cells with either Wnt-3, ΔN89β-catenin, or the were for which is of the Cox-2 with the Cox-2 protein expression levels Wnt-3-expressing cells secreted more compared with the control cells and cells data clearly show that Wnt-3 regulates Cox-2 and periostin expression levels in C57MG a pathway that not involve β-catenin. of the Wnt-3 signaling pathway is GSK-3. whether an of GSK-3 L. Curr. Biol. 1996; Full Text Full Text PDF PubMed Google Scholar), Cox-2 and periostin expression in response to GSK-3 of C57MG cells with of in a increase of Cox-2 protein after h The same that mediated increased Cox-2 levels in a 50% reduced kinase in an in 5 is to the inhibition of GSK-3 after is removed the wash and the inhibition of the GSK-3 is the of of cells with for h in a of periostin mRNA levels 5 data that the of periostin mRNA is regulated at the level of GSK-3, but to this To further that Wnt-3 and the inhibition of GSK-3 but not β-catenin can regulate periostin and antisense β-catenin were to β-catenin expression cells with a of β-catenin mRNA levels by and Cox-2 and periostin levels can be regulated by to the same as cells or cells with the reverse control However, cells with the same of β-catenin mRNA a in gene when treated with medium or for h compared to cells with the β-catenin reverse control The RNA expression levels of β-catenin are shown in B. the periostin and Cox-2 mRNA levels regulated by Wnt-3 and are independent of β-catenin antisense oligonucleotide transfection in periostin mRNA levels in response to is independent of reduced levels of β-catenin. Total RNA from cells that were with β-catenin antisense or reverse control and treated with 30 mm for h was and mRNA levels of β-catenin and periostin were in a PCR and β-catenin mRNA levels were to actin mRNA in each reverse image antisense oligonucleotide transfection gene in response to and C57MG cells were with antisense or reverse control and h treated with medium or 30 mm for The compared with control of the is The β-catenin mRNA levels are by PCR and to mRNA levels in each probe reverse image regulates periostin and Cox-2 mRNA levels independent of β-catenin mRNA Wnt-3 cells and control cells were with antisense or reverse control Total RNA from the cells was h after and β-catenin, and periostin mRNA levels were determined by PCR and to actin or image To this is the first time that in β-catenin levels using antisense inhibition of transcriptional in response to Wnt and has the of a of Wnt target genes expression may be independent of β-catenin. The evidence that Cox-2 and periostin are two genes expression is regulated by Wnt-3 in a pathway that is independent of transcription. have shown that Cox-2 and periostin are regulated by the inhibition of GSK-3 by but not by β-catenin. that a of Wnt target genes that is independent of β-catenin to have shown that β-catenin stabilization to be for data are with studies that mutations in β-catenin, in human and cancer cell result in a B. P. M. E. P. Science. 1997; PubMed Scopus Google Scholar, P.J. Sparks A.B. H. Vogelstein B. Kinzler K.W. Science. 1997; PubMed Scopus Google Scholar). β-catenin then with the transcription and gene transcription. Interestingly, there is in the of C57MG cells when are with or with in to by Wnt-3, which a 1 A and data not in that even as there is genes regulated by Wnt and by β-catenin as as by inhibition of GSK-3, there also be the a differential display was used to the gene profiles regulated by Cox-2 expression increased and periostin expression in response to of the Wnt genes were also regulated by inhibition of GSK-3 but overexpression of to the same to overexpression of ΔN89β-catenin, the of β-catenin expression levels by antisense oligonucleotide transfection was a used to the of the Wnt pathway downstream of GSK-3. The regulation of Cox-2 and periostin by Wnt-3 and was not by the of β-catenin expression levels and data a of genes downstream of GSK-3 that is regulated by Wnt-3 in a GSK-3 and can Wnt in J. Lee P.S. Dev. Biol. 1997; PubMed Scopus Google Scholar) and in the gene the of the gene is by the of β-catenin antisense Wnt-3 and inhibition of GSK-3 by the mRNA levels of periostin. 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