Conceived and designed the experiments: AKF CMF SWWB. Performed the experiments: AKF YSB. Analyzed the data: AKF YSB SWWB. Contributed reagents/materials/analysis tools: AKF YSB CMF. Wrote the paper: AKF SWWB.
The authors have declared that no competing interests exist.
The cell-cell adhesion molecule cadherin-11 is important in embryogenesis and bone morphogenesis, invasion of cancer cells, lymphangiogenesis, homing of cancer cells to bone, and rheumatoid arthritis. However, very little is known about the regulation of cadherin-11 expression.
Here we show that cell density and GSK-3β regulate cadherin-11 levels in cancer cells. Inactivation of GSK3β with lithium chloride or the GSK3 inhibitor BIO and GSK3β knockdown with siRNA repressed cadherin-11 mRNA and protein levels. RNA Polymerase II chromatin immunoprecipitation experiments showed that inhibition of GSK3 does not affect cadherin-11 gene transcription. Although the cadherin-11 3′UTR contains putative microRNA target sites and is regulated by Dicer, its stability is not regulated by GSK3 inhibition or density. Our data show that GSK3β regulates cadherin-11 expression in two ways: first a β-catenin-independent regulation of cadherin-11 steady state mRNA levels, and second a β-catenin-dependent effect on cadherin-11 3′UTR stability and protein translation.
Cadherin-11 mRNA and protein levels are regulated by the activity of GSK3β and a significant degree of this regulation is exerted by the GSK3 target, β-catenin, at the level of the cadherin-11 3′UTR
Cadherin-11, also known as OB-cadherin was first identified in mouse osteoblasts and is normally expressed in cells with a mesenchymal phenotype and of mesodermal origin, including the mesenchyme of the kidney and brain during development
Little is known about the regulation of cadherin-11 expression. TGFβ1 regulates cadherin-11 in cultured extravillous cytotrophoblasts, and progesterone, but not 17-beta-estradiol, regulates cadherin-11 in cultured endometrial stromal cells undergoing decidualization
The principle pathway associated with canonical Wnt signaling involves inhibition of GSK3. GSK3 is intricately involved in many pathways essential to cellular function. Other mechanisms that also negatively regulate GSK3β include GSK3β phosphorylation on Ser9 by Akt and inhibition by LiCl treatment and 6-bromoindirubin-3′-oxime (BIO) treatment
In this study we use breast cancer cells with low levels of endogenous activated GSK-3 and β-catenin and prostate cancer cells with constitutively activated GSK-3 and β-catenin to show that, GSK3β regulates cadherin-11 expression in two ways. The first mechanism is through β-catenin-independent regulation of steady state cadherin-11 mRNA levels. The second mechanism involves a β-catenin-dependent effect on cadherin-11 3′UTR stability and protein translation.
MDA-MB-231 cells, a mesenchymal-like breast cancer cell line that expresses cadherin-11, have undetectable levels of other cadherins and low levels of activated β-catenin. In pilot experiments we noticed that cadherin-11 expression varied noticeably depending on the number of cells plated.
A and B: MDA-MB-231 cells were plated at two densities, 50% and 90% confluency, called low and high, respectively. The cells were allowed to grow overnight in serum-free medium. 24 hours after plating cells were treated with 20 mM NaCl (N) or 20 mM LiCl (L). 24 hours after treatment, RNA and protein were collected for real-time PCR (A) and Western (B) analysis. C: Cancer cells BT549, Hs578T, PC-3, and Kato-III were plated at a medium density and allowed to adhere. Cells were then treated with 20 mM LiCl or NaCl control. 48 hours after transfection protein was collected for Western blot analysis. D: MDA-MB-231 cells were plated at a medium density, serum starved overnight, and treated with 20 mM NaCl or 20 mM LiCl. 24 hours after treatment, the cells were washed once with PBS and maintained in serum-free medium containing only 20 mM NaCl. RNA was collected at the times indicated for real-time PCR analysis. E and F: MDA-MB-231 cells were plated at a medium density and serum starved overnight. 16 hours after plating, the cells were treated with 1 μM meBIO (control), 1 μM BIO, or 20 mM LiCl. RNA (E) or protein (F) was collected at the designated times and analyzed using real-time PCR (E) and Western blot analysis (F). G: MDA-MB-231 and PC-3 cells were transfected with either non-specific scrambled siRNA (siScramble), or siRNA directed against GSK3β (si GSK3). 48 hours after transfection protein was isolated for Western blot analysis.
To test if GSK3β regulates cadherin-11 expression at the level of transcription, MDA-MB-231 cells were treated with the RNA polymerase II inhibitor, actinomycin D and were compared to the results of cells treated with LiCl.
A: MDA-MB-231 cells were grown at a medium density for 16 hours in the absence of serum. The cells were then pretreated with 5 μg/ml actinomycin D or an equivalent volume of ethanol (untreated). 30 minutes later the cells were treated with either 20 mM NaCl (control) or LiCl. At the indicated time points, RNA and protein were collect for real-time PCR analysis. B: MDA-MB-231 cells were treated with 20 mM NaCl or LiCl for 24 hours. Genomic DNA was then harvested for RNA polymerase II ChIP analysis followed by PCR specific to GAPDH and cadherin-11.
In many situations regulation of mRNA stability and its capacity for translation are mediated by the 3′-untranslated region (UTR). If the cadherin-11 3′-UTR is important in the normal regulation of cadherin-11 expression we might expect it to be conserved across species.
A: Sequence of cadherin-11 3′-UTR according to the Ensembl database (NM_001797). Bolded sequences indicate the poly-A signals and site respectively. Blue highlighted sequences indicate Shaw-Kamens, destabilizing sequences. The first two red highlighted sequences indicate the primers used to design pGL3-CDH11-3′UTR NCBI, as denoted by the bracket. The first and last red highlighted sequences indicate the primers used to design pGL3-CDH11-3′UTR Ensembl, as denoted by the bracket. B: Predicted secondary structure of the Ensembl cadherin-11 3′UTR (as predicted by GeneBee). C: Predicted secondary structure of the Ensembl E-cadher 3′UTR (as predicted by GeneBee). D: RT-PCR of PC3 RNA using primers designed approximately every 500 bp.
ClustalW Nucleotide Sequence Alignment compared to |
||||||
Species | E-cadherin protein coding sequence | E-cadherin 3′UTR | Cadherin-11 protein coding sequence | Cadherin-11 5′UTR | Cadherin-11 3′UTR (NCBI) | Cadherin-11 3′UTR (Ensembl) |
79 | 17 | 89 | 84 | 94 | 85 | |
80 | 27 | 90 | 76 | 94 | 84 | |
99 | 98 | 99 | 98 | 99 | 99 | |
64 | N/A | 80 | 59 | 86 | 68 |
The NCBI database identifies a region of the cadherin 3′UTR which spans approximately 1 kb and includes a traditional poly-A signal and site. However, the Ensembl database identifies a cadherin-11 3′UTR of roughly 3 kb which includes a second poly A signal 2363 bp from the stop codon (
To investigate the function of the cadherin-11 3′-UTR, it was cloned from MDA-MB-231 genomic DNA. A series of stop codons were included at the 5′-end of the construct followed by the DNA sequence directly 3′ to exon 13, the 3′-UTR. Both the NCBI and Ensembl 3′UTRs were cloned using the red highlighted primer sequences in
In cadherin-11 positive PC-3 and MDA-231 cells both luc-CDH11 3′UTR NCBI and luc-CDH11 3′UTR Ensembl were significantly less active than pGL3, which has the SV40 poly A addition signal and site (
A: PC-3, MDA-MB-231, and HEK 293 cells were transfected with pGL3-Promoter, pGL3-CDH11-3′UTR NCBI, or pGL3-CDH11-3′UTR Ensembl and pCMV-Renilla. 48 hours after transfection, cells were lysed using passive lysis buffer and luciferase activity was analyzed. (* indicates a p-value >0.01; ** indicates a p-value >0.001) B: MDA-MB-231 and PC-3 cells were transfected with pGL3-CDH11 3′-UTR and pCMV-Renilla along with either non-specfic siRNA (siScramble) or siRNA directed against Dicer (siDicer). 48 hours after transfection, cells were lysed using passive lysis buffer and luciferase activity was analyzed. (* indicates a p-value >0.05) C and D: MDA-MB-231 cells were transfected with non-specific siRNA (siScramble), or siRNA directed against Dicer (si Dicer). Cells were collected for real-time PCR (C) and Western blot (D) analysis 72 hours after transfection.
To investigate if microRNA influenced the activity of luc-CDH11 3′-UTR we knocked down Dicer, which is essential for the production of microRNAs. Removal of Dicer significantly increased the activity of luc-CDH11 3′-UTR indicating that microRNAs regulate the endogenous activity of the cadherin-11 3′-UTR (
Perhaps the best known substrate of GSK3β is β-catenin and many studies have shown that Wnt-mediated inhibition of GSK3β stabilizes and activates cytoplasmic β-catenin
A: PC-3 cells were transfected with pGL3-CDH11-3′UTR NCBI or pGL3-CDH11-3′UTR Ensembl and pCMV-Renilla and along with either non-specific siRNA or siRNA directed against CTNNB1. 48 hours after transfection cells were were lysed using passive lysis buffer and luciferase activity was analyzed. Luciferase activity was normalized to renilla activity. (** indicates a p-value >0.001). B: MDA-MB-231 and PC-3 cells were transfected with either non-specific scrambled siRNA (siScramble) or siRNA directed against CTNNB1 (si β-cat). 24 hours after transfection cells were treated with 1 μM BIO or meBIO (control). 48 hours after transfection protein was isolated for Western blot analysis. C: MDA-MB-231 and PC-3 cells were transfected with either non-specific scrambled siRNA (siScramble) or siRNA directed against CTNNB1 (si β-cat). 24 hours after transfection cells were treated with 1 μM BIO or meBIO (control). 48 hours after transfection RNA was isolated for real-time PCR analysis. (* indicates a p-value >0.05; ** indicates a p-value >0.005)
The majority of patients dying from breast or prostate cancer have metastases to the skeleton. Although bone metastases are incurable, patients survive several years suffering serious morbidity, including fractures, spinal cord compression, severe bone pain, and hypercalcemia
The best studied mechanism by which GSK3β is regulated is Akt-dependent serine phosphorylation. Growth factors, such as insulin, activate the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. PI3K converts PIP2 to PIP3, a critical step necessary for the activation of PKB/Akt. PTEN is a phosphatase commonly mutated in cancers that is necessary for the dephosphorylation of PIP3 thereby inhibiting Akt activation. Once activated Akt acts on many cellular proteins including mTOR, caspase 9, Bad, IKK, and GSK3β. Akt phosphorylates GSK3β on serine 9 and GSK3α on serine 21 to inhibit their kinase activity. Understanding GSK3β regulation by the Akt pathway is important to the present study because a PTEN mutation in PC-3 prostate cancer cells results in activated Akt, GSK3β inhibition, and activation of endogenous β-catenin
LiCl (L9650), NaCl (S3014), and actinomycin D (A1410) were obtained from Sigma-Aldrich (Germany), meBIO (361556) and BIO (361550) from Calbiochem (San Diego, CA), anti-cadherin-11 (5B2H5) from Invitrogen (32-1700, Carlsbad, CA), anti-β-catenin antibody from BD Biosciences (610154, San Jose, CA), Dicer antibody from Abcam (ab14601, Cambridge, MA), anti-GSK3β antibody from Cell Signaling Technologies (9315, Boston, MA), anti-FLAG (M2) was from Sigma (F3165, Germany), anti-GAPDH was from Research Diagnostics Inc (TRK5G4-6C5, Flanders, NJ), anti-Snail (H-130) (sc-28199) antibody from Santa Cruz. The anti-eIF6 antibody S13 was a generous gift from Dr. Biffo and generated against a C-terminus peptide of eIF6
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Plasmid DNA encoding wild type (WT) β-catenin was used as previously described and DNA encoding wild type Snail was a kind gift from Dr. Mien-Chie Hung
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MDA-MB-231, Hs578T, BT549 breast cancer cells, PC-3 prostate cancer cells, and HEK293 cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 5% fetal bovine serum (FBS) in 5% CO2 incubator at 37°C. All transient transfections of plasmid DNA and siRNA in MDA-MB-231 and PC-3 cells were performed with Amaxa electroporation system (Amaxa, Inc, Geithersburg, MD) according to the manufacturer's protocol. Transient transfections for immunofluorescence were preformed using Lipofectamine 2000 Transfection Reagent (11668-019, Invitrogen, Carlsbad, CA) and luciferase analysis using Lipofectamine 2000 (Invitrogen) for PC-3 cells, ProFection Mammalian Transfection System—Calcium Phosphate (E1200, Promega, Madison, WI) for MDA-MB-231 cells, and FuGENE 6 Transfection Reagent (11814443001, Roche, Switzerland) for HEK293 cells.
MDA-MB-231 or PC-3 cells were incubated in the presence or absence of LiCl (20mM) or BIO (1 μM) for 24 h. RNA was isolated using Trizol (15596-018, Invitrogen) combined with RNAeasy (74106, Qiagen, Valencia, CA) according to the manufacturer's instructions.
MDA-MB-231 cells incubated in the presence or absence of 20 mM LiCl for 24 hours. Total RNA was isolated using the miRVana RNA isolation kit (AM1562, Ambion).
Relative quantitation was used to evaluate the raw data obtained from real-time PCR (7900 HT real time PCR system, Applied Biosystems, Foster City, CA). Single-stranded cDNA was prepared using TaqMan Reverse Transcription Reagents (N808-0234, Applied Biosystems) following the manufacturer's protocol. TaqMan Universal PCR Master Mix (4304437, Applied Biosystems) was used for all reactions. All primer/probe mixes (CDH11 Hs00156438_m1, GAPDH Hs99999905_m1) were obtained from Applied Biosystems and performed in triplicate. The samples were analyzed using the delta-delta Ct method of analysis
cDNA was generated by TaqMan MicroRNA Reverse Transcription kit (4366596, Applied Bioscience) following manufacturer's protocol. Primer/probe mixes specific for each microRNA (RNU6B, 4373381; hsa-miR-19a, 4373099; hsa-miR-27a, 4373287; hsa-miR-33, 4373048; hsa-miR-101, 4373159; hsa-miR-133b, 4373172; hsa-miR-337, 4373044; hsa-miR-424, 4373201) were obtained from Applied Biosystems (Foster City, CA). TaqMan Universal Master PCR Mix (4304437, Applied Biosystems) was used for all reactions. All experiments were performed in triplicate. The average value of the triplicate readings for each unknown was normalized to the corresponding value for U6 RNA. The samples were analyzed using the delta-delta Ct method of analysis
One 15 cm tissue culture dishes were plated with 95% confluent MDA-MB-231 cells for each condition. Cells were treated with 5 μg/ml actinomycin D for 30 minutes in serum-free DMEM. As specified, cells were incubated with 20 mM NaCl or LiCl for an additional 24 hours. 37% formaldehyde solution was added to each plate for a final concentration of 1.5% and incubated at 37°C for 15 minutes. Plates were washed one time with PBS containing 0.125 M glycine and Complete Mini Protease Inhibitor Cocktail (11836153001, Roche), and then a second time with PBS plus inhibitor. Cells were collected and spun for at 2000 rpm, 4°C for 5 minutes. The pellet was resuspended in 1 ml SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.1) with protease inhibitors. Cells were then sonicated using a 15 second on, 45 second off program 4 times consecutively; then diluted 1:10 in ChIP Dilution Buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH .1, 167 mM NaCl) plus protease inhibitor. The samples were then precleared overnight at 4°C with 75 μl Protein A/G Plus-agarose beads (sc-2003, Santa Cruz) supplemented with 3 μl 10 mg/ml Sonicated Salmon Sperm DNA (201190, Stratagene, La Jolla, CA) and 13 μl 1 mg/ml BSA. Samples were then incubated with 10 μg RNA Polymerase II (N-20) (sc-899, Santa Cruz) overnight, tumbling at 4°C. Add 40 μl Protein A/G Plus-agarose beads for 2 hours rotating at 4°C. Samples were spun at 1000 rpm for 1 minute, then washed once with each: Low Salt Immune Complex Wash Buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 150 mM NaCl), High Salt Immune Complex Wash Buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 500 mM NaCl), LiCl Immune Complex Wash Buffer (0.25 M LiCl, 1% Nonidet P-40, 1% deoxycholic acid, 1 mM EDTA, 10 mM Tris-HCl, pH 8.1), and TE buffer. Samples were eluted twice in 100 μl in elution buffer (1% SDS, 0.1 M NaHCO3) vortexing for 15 minutes at room temperature. 12 μl 5 M NaCl was added to eluate and incubated at 65°C overnight. Then 4 μl 0.5 M EDTA, 8 μl 1M Tris-HCl, pH 6.5 and 2 μl of 10 mg/ml proteinase K was added and incubated at 45°C for 1 hour. The samples were cleaned up using the QIAGEN PCR Clean up kit (QIAGEN). PCR was performed using TaKaRa Premix Ex Taq kit (RR039, TaKaRa, Otsu, Shiga, Japan). The final reaction contains: 1× Premix Ex Taq, 2.5 μM of each primer, 8% DMSO, and 20% processed DNA (or 20% of a 1:10 dilution of input). For amplification of GAPDH, forward: (
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reverse: (
Cells were treated for the indicated times. Cells were rinsed once with PBS and lysed with sample buffer (2% SDS, 10% glycerol, 10 mM Tris, pH 7.5) containing 1 mM sodium orthovanadate, 0.05 M sodium fluoride, and Complete mini protease inhibitors (11836153001, Roche Applied Science, Indianapolis, IN). Cell lysates were boiled for 10 minutes. Protein concentration was determined with a Bio-Rad DC Protein Assay (500-0116, Bio-Rad, Hercules, CA). After SDS-poly acrylamide gel electrophoresis, proteins were transferred to Protran BA 83 Nitrocellulose (10402495, Germany). Membranes were blocked with 5% milk in Tris-Buffered Saline containing 0.1% Tween-20, and incubated with primary antibody overnight at 4°C and subsequently with HRP-labeled secondary antibody. Proteins were visualized with ECL chemiluminescent reagents (RPN2106, Amersham Biosciences, Piscataway, NJ) or SuperSignal West Femto (34095, Pierce biotechnology Inc., Rockford, IL) using X-ray films (Denville Scientific Inc., Metuchen, NJ).
Cells were plated at 2×105 in a 12-well plate in growth medium. Cells were transfected in triplicate with 1.8 ug of
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The authors wish to thank Robert Lechleider for his advice and Becky Hoxter for her technical expertise and acknowledge the support the following LCCC Core Facilities (NIH P30 CA51008): microscopy, tissue culture, macromolecular analysis and microarray.