Proteasome Down-regulation is Partly Mediated by ... - Springer Link

J Korean Soc Appl Biol Chem (2013) 56, 157−163 DOI 10.1007/s13765-013-3004-1

ORIGINAL ARTICLE

Proteasome Down-regulation is Partly Mediated by Slug/Snail2 in Hepatocarcinoma Cells Jin Young Kim · Yeon-Ki Kim · Young Mee Kim · Seogjae Lee · Sanggyu Park · Baek Hie Nahm · Dong-Sun Lee · Moonjae Cho

Received: 4 January 2013 / Accepted: 30 January 2013 / Published Online: 30 April 2013 © The Korean Society for Applied Biological Chemistry and Springer 2013

Abstract Snail family proteins (Snail1 and Slug/Snail2) are transcription factors that regulate transcription of molecules during epithelial-mesenchymal transition (EMT). Snail1/2 is known to bind to the E-box motif (CANNTG). The proteasome activity is decreased in EMT (Kim et al., 2011), and several E-box motifs are found in the promoters of genes coding for proteasome subunits. We used a new protein-binding microarray to specify the Slug/Snail2 binding sequence. Among 563 9-mer clusters, the motif CACCTGC yielded the highest P-value in the WilcoxonMann-Whitney test. Within this motif, the A and T were absolutely required, and CC was preferred, but could be replaced by GG with little effect. In hepatocytes overexpressing Slug/

J. Y. Kim Present address: Jeju special self-governing province Agricultural Research & Extension Services, Seogwipo, Jeju 697-827, Republic of Korea J. Y. Kim · Y. M. Kim · S. Lee · M. Cho () Department of medicine , School of Medicine, Jeju National University, Jeju 690-756, Republic of Korea E-mail: [email protected] Y. -K. Kim · B. H. Nahm GreenGene Biotech Inc., Myongji University, Yongin 449-728, Republic of Korea S. Park Division of Life & Environmental Science, Daegu University, Daegu 712714, Republic of Korea B. H. Nahm Division of Bioscience and Bioinformatics, Myongji University, Yongin 449-728, Republic of Korea D.-S. Lee College of Applied Life Science (RISA), Jeju National University, Jeju 690-756, Republic of Korea M. Cho Institute of Medical Science, Jeju National University, Jeju 690-756, Republic of Korea

Snail2, the 20S proteasome expression and proteasome activity were decreased partly due to the down-regulation of proteasome subunit beta type 2 (PSMB2) and PSMB3 transcription. Keywords E-box motif · epitherial-mesenchymal transition · protein-binding microarray · Slug · Snail

Introduction The Snail family proteins, such as Snail, Slug/Snail2, EF1 (ZEB1), SIP1 (ZEB-2), and TWIST, contain four tandem C2H2 zinc finger motifs at the COOH-terminus and a highly conserved SNAG repression domain (1–9 amino acids) that is important for co-repressor interaction at the NH2-terminus (Shih and Yang, 2011). The zinc finger binds to a DNA target sequence called the E-box motif (CANNTG), which is usually found in tandem in Snail target genes, including E-cadherin (Comijn et al., 2001). Slug/Snail2, which belongs to the highly conserved Slug/Snail family, is found in diverse species from Caenorhabditis elegans to humans. In vertebrates, the Slug/Snail2 transcription factors have been proposed to participate in developmental epithelial-mesenchymal transition (EMT) such as neural crest cell migration (Vernon and LaBonne, 2006) as well as in pathological EMT in various cancers including ovarian cancer, breast cancer, and melanoma (Kurrey et al., 2005; Perez-Mancera et al., 2005; Vuoriluoto et al., 2011). Stability of Slug/Snail2 is regulated by ubiquitin-mediated proteasome degradation (Vernon and LaBonne, 2006) and glycogen synthase kinase 3-beta activity (Kim et al., 2012). In eukaryotes, the ubiquitin-proteasome degradation via the 26S proteasome multimeric protein complex is the main mechanism of protein degradation and is critical for signal transduction, transcriptional regulation, response to stress, and control of receptor function (Adams et al., 2000). The transforming growth factor β1

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(TGF-β1) signaling pathway is involved in EMT (Han et al., 2005), and the transcriptional activation by the TGF-β1 pathway is regulated by the ubiquitin-proteasome pathways (Zhang and Laiho, 2003), thus providing increasing evidence of the link between proteasome activity and EMT. Up-regulation of the proteasome catalytic subunit during neuronal differentiation was reported by Klimaschewski et al. (2006). However, data in the literature are very limited regarding the transcriptional/translational regulation of each proteasome subunit in vertebrates. In Saccharomyces cerevisiae, it has been established that the transcription factor RPN4 mediates expression of the proteasome subunit genes by binding to their promoters via a conserved motif (5-GGTGGCAAA-3), which is named the proteasome-associated control element sequence (Mannhaupt et al., 1999). The expression of the ATPase subunit 4 of the 26S proteasome is regulated by heregulin-β1 in breast cancer cells (Barnes et al., 2005), and nuclear factor erythroid-derived 2-related factor 1 (Nrf1) induces the transcription of proteasome genes in mouse embryonic fibroblasts (Radhakrishnan et al., 2010). Transcription factors interact with specific DNA sequences to control gene expression. The DNA-binding properties of proteins have been investigated by traditional methods, such as the electrophoretic mobility shift assay and filter-binding assay (Garner and Revzin, 1981; Soderman and Reichard, 1986). However, these methods can be effectively used only when prior knowledge of the DNA sequence is obtained via promoter reporter assays. With the availability of whole-genome sequences and advances in microarray technology, comprehensive genome-wide methods have been developed to characterize protein-DNA binding specificities (Schena et al., 1995). In the present study, we used a new quadruple 9-mer-based protein-binding microarray to find the specific binding sequence of Slug/Snail2. We found that the promoter of the proteasome subunit gene contained Slug/Snail2 putative binding sequences, and that overexpression of Slug/Snail2 decreased the proteasome expression and activity.

Materials and Methods Antibodies and reagents. Monoclonal antibodies against proteasome 19S and proteasome subunit beta type 2 (PSMB2) and polyclonal antibodies against proteasome 20S were purchased from Abcam (UK). Monoclonal glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was purchased from Cell Signaling Technology (USA). Mouse anti-tubulin-alpha antibody was purchased from BioLegend (USA). Proteasome inhibitor MG132 was purchased from Calbiochem (USA). Proteasome activity assay kit was purchased from Promega (ProteasomeGlo™ Chymotrypsin-Like Cell-Based Reagent, USA). Protein-binding microarray (PBM). We designed a PBM, which we refer to as Q9-PBM, in such a way that the target probes are synthesized as quadruples of all possible 9-mer combinations.

J Korean Soc Appl Biol Chem (2013) 56, 157−163

A total of 131,072 features were selected after consideration of the reverse complimentary sequences of all 9-mer combinations, and 101,073 features were replicated to confirm the binding consistency. Each 9-mer was quadrupled and linked to a polymerase chain reaction primer-binding site following five thymidine linkers attached to the slide. These repetitive thymidine sequences provide highly consistent results by which consensus binding motifs can be extracted, thereby allowing unequivocal interpretation. The microarray was manufactured by Agilent Technologies (USA), and the reverse complementary DNA strand of each probe was synthesized on the slide via thermo-stable DNA polymerase. Slug/Snail2 was expressed in E.coli as a fusion protein, with its N-terminal fused to DsRed fluorescent protein. The resulting microarray was scanned, and red spots through the microarray suggested that reverse complementary strands were successfully synthesized. The DsRed-fused DNA-binding protein was applied to the double-stranded Q9-PBM, and the fluorescence intensity of the bound protein was acquired using a microarray scanner (Molecular Devices, USA). The consensus binding sequence was determined based on signal strength. In general, the rank-ordered signal distribution of the bound protein showed a deep leftward slope followed by a heavy right tail (Fig. 1A), as observed in a previous report (Jung et al., 2012). Because the probes in the deep slope region differed by only one base, we assumed that the signal distribution was due to a specific interaction between the protein and features on the microarray. Two independent linear models, y=ax+b, were applied to the deep and the heavy right-tail region using R statistical language. The spot intensities were rank-ordered, and enrichment scores of 5-, 6-, and 7-mers were determined. Spots that exhibited strong intensity and high enrichment were subjected to alignment. The sequence logo was plotted using the public software seqLOGO (http://www.bioinf.ebc.ee/EP/EP/ SEQLOGO/). Cell culture and transfection. The human hepatocarcinoma cells, SNU449 (Korean Cell Bank, Korea), were cultured in RPMI medium with 10% fetal bovine serum (Gibco BRL, USA), and the human hepatoma cells, Huh-7 (Korean Cell Bank, Korea), were cultured in Dulbeccos’ modified Eagle’s medium with 10% fetal bovine serum (Gibco BRL). Huh7 and SNU449 cells transfected with pcDNA3-Slug/Snail2 were selected by G418 (Geneticin®, AG Scientific, USA) after transfection using Lipofectamine 2000TM (Invitrogen, USA) according to the manufacturer’s protocol. Proteasome activity assay. Reagent treatments were performed in each growth medium containing 5% FBS. A total of 500 µL of cells (1×105 cells/mL) were seeded in 24-well plates and transfected with pcDNA3-Slug/Snail2, as described above. After trypsinization, cells were (1×104 cells/mL) were seeded in a 96-well plate (100 µL/well). Proteasome-Glo™ Chymotrypsin-Like Cell-Based Reagent (Promega, USA) was added at a volume of 100 µL/well and incubated for 5 min. Luminescence was measured using a luminometer (FLUOstar Optima, BMG Labtech, UK). Assays were conducted in triplicate. Immunoblot analysis. For immunoblotting, cells were washed


with phosphate-buffered saline (PBS) and harvested in RIPA buffer [50 mM Tris-HCl pH 8.1, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS)]. Total protein concentration was measured using the bicinchoninic acid assay (Pierce Chemical Co., USA). Samples were loaded and separated on 12% SDS-polyacrylamide gels, transferred onto polyvinylidene fluoride membranes, and probed with specific primary antibodies. Signals were detected using enhanced chemiluminescent substrate (WEST-ZOL, iNtRON Biotechnology Inc., Korea). Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Total RNA was isolated from the cell lines using the TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. Reverse transcription was carried out using the Reverse Transcription System kit from Promega. PCR primers for amplification of PSMB1 were: Forward 5'-GCAGCCGTGCGAT GTTGTCC-3', Reverse 5'-GAGCACACAGATCAGTCCTTCC-3'; PSMB2: Forward 5'-TCGTGCTGTGTCGGACCTGC-3', Reverse 5'-GTTCCCTGGCAAGTGGGAGG-3'; PSMB3: Forward 5'-GA GGGGTCCTAGTACACCGC-3', Reverse 5'-CAGGGTTAGTCC ATTCGGGC-3'; PSMB4: Forward 5'-GCTACCGTGACTAAGA TGGAAGC-3', Reverse 5'-TCAAAGCCACTGATCATGTGGG C-3'; GAPDH: Forward 5'-GAAGGTGAAGGTCGGAGTC-3', Reverse 5'-GAAGATGGTGATGGGATTTC-3'; β-actin: Forward 5'-CTTCCTGGGCATGGAGTC-3', Reverse 5'-GCCAGGGTAC ATGGTGGT-3'; Slug/Snail2: Forward 5'-CCCGTTAACATGCCG CGCTCTTTC-3', Reverse 5'-TTTCTCGAGTCAGCGGGGACA TCC-3'. PCR was performed using Taq polymerase (iNtRON Biotechnology Inc.). PCR was initiated by heating the samples for 5 min at 95oC, followed by denaturation at 95oC for 1 min, annealing at 60oC for 1 min (30 cycles), and elongation at 72oC for 1 min. For amplification, the PCR conditions were as follows: 5 min at 95oC, followed by 35 cycles of 95oC for 1 min, 55oC for 1 min, and 72oC for 1 min. Samples were analyzed by electrophoresis on 1% agarose gels containing 0.002% nucleic acidstaining solution (RedSafeTM, iNtRON Biotechnology Inc.). Immunofluorescence microscopy. Cells were seeded in well chamber slides. Cells were washed with cold phosphate buffered saline (PBS), fixed in 3.7% formaldehyde in PBS for 10 min, permeabilized with 0.5% Triton X-100 in PBS for 10 min at room temperature, and washed three times with PBS for 10 min. Cells were blocked with 5% bovine serum albumin, incubated with primary antibody for 1 h (anti-proteasome 20S; dilution 1:200) and washed with PBS three times. They were incubated with secondary antibody-conjugated tetramethylrhodamine isothiocyanate (Chemicon, USA) in dark for 1 h and washed three times with PBS for 10 min. Cells were mounted in a mounting solution (DakoCytomation, Denmark) and examined by fluorescence microscopy. Statistical analyses. All experiments were performed independently three times unless otherwise indicated. Data were presented as means ± standard deviation. Data were analyzed using the Sigma plot t-test.

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Fig. 1 Schematic representation of Slug binding microarray analysis. (A) Rank analysis of Slug PBM binding. Based on the rank-ordered signal distribution, two independent linear models, y=ax+b, were applied in the steep (b1=55538.1, slope= −31.1) and the heavy right (b1=5703.4, slope= −0.0036) tail regions. (B) Total position weight matrix. Spots that exhibited strong intensity and high enrichment were subjected to alignment. (C) Sequence logo of the determined consensus binding sequence of Slug.

Results We reported previously that the proteasomal activity of SNU449 and PLC/PRF5 hepatocytes decreased after hepatocyte growth factor treatment, and that the expression of cell-cell adhesion molecules was clearly reduced after MG-132 treatment (Kim et al. 2011). In hepatocytes, we found that TM4SF5 expression results in EMT (Lee et al., 2008) and enhances stability of proteins (Kim et al., 2011) via a decrease in the transcriptional level of proteasome subunit genes. Transfection with TM4SF5 did not increase protein levels of Snail1 (Lee et al., 2008). Because the degradation of Slug/Snail2 depends on the ubiquitin-proteasome pathway mediated by murine double minute-2 (Wang et al., 2009), we examined whether Slug/Snail2 could regulate the transcription of proteasome genes. Extraction of Slug/Snail2-binding motif. Slug/Snail2-DSRed

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Fig. 2 Distribution of the E- box sequences (CACCTG, CAGGTG, CAGCTG, CACGTG) in human 20S proteasome genes. The promoter sequences located ~3000 bp upstream from the annotated transcription initiation sites of known human 20S proteasome genes in databases were analyzed. The PSMA (A) and PSMB (B) genes containing the E-box sequences and the relative positions of the E-boxes are presented. (Each color arrow indicates a specific sequence.

fusion proteins were reacted with a Q9-PBM to search for the exact binding motifs. The rank-ordered signal distribution of the SLUG_100809 PBM showed a deep leftward slope followed by a heavy right tail, because the signal distribution was due to a specific interaction between the protein and features on the microarray. Two independent linear models, y=ax+b, were applied in the deep (b1=55538.1, slope= −31.1) and the heavy right (b1= 5703.4, slope= −0.0036) tail regions. Spots with intensities higher than 5,703 were used. In total, 14,923 ninemers are clustered to give motifs (Fig. 1A). From the deep left region, ninemers were clustered based on best alignment using the highest ninemers among the clusters as seeds. The website gives an intensity profiling figure, sequence logos, and their related statistics. Among the clusters, 563 9-mers were clustered to give the motif CACCTGC, and its position weight matrix (PWM) was obtained. A Wilcoxon-Mann-Whitney test for CACCTGC yielded a p-value of 4.40e-08 (Fig. 1B and 1C). Promoter of the proteasome subunit contains putative Slug/ Snail2-binding sequence. Because the proteasome activity was down-regulated by EMT, and Snail and Slug are the typical EMTrelated repressors, promoter analysis was performed for proteasome subunit genes for E-box (CACCTG, CAGGTG, CAGCTG, CACGTG). The proteasome consists of 20S and 19S complexes, and the 20S complex contains seven alpha (PSMA1–7) and seven beta (PSMB1–7) subunits. Promoters of all the 20S subunit genes contain many putative Slug-binding sequences (Fig. 2). Because

the 20S complex consists of 28 subunits containing two of each PSMA and PSMB genes (α7β7β7α7), it is possible that regulating the synthesis of only one subunit can control the activity of the whole complex. Among the PSMA genes, PSMA3, 4 and 6 contain a cluster of E-box sequences (Fig. 2A), and among the PSMB genes, PSMB2, 3, and 5 contain a cluster of E-box sequences (Fig. 2B). Slug/Snail2 expression decreases proteasome activity via repression of PSMB2 and PSMB3 transcription. To investigate whether the Slug/Snail2 protein can regulate the transcription of 20S proteasome subunits, we transfected the Slug/Snail2 gene in hepatoma cells. Expression of PSMB genes was measured by RTPCR using gene-specific primers. Among the seven PSMB genes, PSMB2 and PSMB3 showed remarkable repression (Fig. 3A). To confirm the repression of the proteasome subunits at the protein level, immunoblotting and immunostaining were performed using anti-S19 and anti-20S antibodies. The over-expression of Slug/ Snail2 down-regulated the PSMB2 and PSMB3 mRNA expression in both SNU449 and Huh7 cells (Fig. 3B). Immunoblot analysis showed decrease in 20S expression (Fig 3B). Immunostaining also showed a decrease in the expression of 20S proteasome following transfection with Slug protein (Fig. 3C). To confirm whether Slug expression inhibits proteasome activity, the hepatocytes were transfected with Slug prior to analysis of proteasome activity. Proteasome activity of Slug-expressing cells usually decreased to 20–30% of mock-transfected cells (Fig. 3D),


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Fig. 3 Slug/Snail2 expression decreases proteasome activity via repression of PSMB2 and PSMB3 transcription. (A) Overexpression of Slug/Snail2 down-regulated the transcription of PSMB genes. A pcDNA3-Slug/Snail2 vector or pcDNA control plasmid was transfected into Huh7 cells. Total mRNA was purified, and RT-PCR was performed. (B and C) Overexpression of Slug/Snail2 decreases the expression of 20S proteasome complex. A pcDNA3-Slug/Snail2 vector or pcDNA control plasmid was transfected into Huh7 or SNU449 cells, and the 20S proteasome complex was analyzed by Western blotting using anti-19S or anti-20S antibodies and by immunostaining using the anti-20S antibody. (D) Slug-mediated down-regulation of proteasome activity. Proteasome activity was analyzed in SNU449 cells stably transfected with mock or Slug. Proteasome activity was measured, as described in the Material and Methods.

indicating that Slug over-expression correlated with downregulate 20S and proteasome activity.

Discussion We previously reported that TM4SF5 expression induced EMT in hepatocytes, and proteasome activity was down-regulated during the EMT process (Kim et al., 2011). In the present study, we provide more details on how the proteasome activity is repressed. A new Q9-PBM that identified the CACCTGC sequence as the Slug putative binding sequence was used. We also found that the promoter of proteasome alpha and beta subunit genes contains a large number of E-box sequences (CACCTG, CAGGTG, CAGCTG, and CACGTG). In hepatocytes overexpressing Slug/Snail2, the 20S proteasome expression and proteasome activity were decreased due to the down-regulation of PSMB2 and PSMB3 transcriptions. Slug, which belongs to the Snail family of proteins, is a typical EMT-related transcription factor containing zinc finger-binding motifs. The sequence ACAGGTG has been reported as a human Slug-binding sequence by Inukai et al. (1999) using PCR-based binding site selection. They also claimed that CA and GT are

critical for binding, and a 39 oligomer contains two direct repeats in the same 5'–3' orientation. The zinc finger binds to a DNA target sequence called the E-box motif (CANNTG) (Comijn et al., 2001). Our results showed that CACCTGC displayed the highest PWM value. followed by CAGGTGC (Fig. 1C). Given that CAGGTG is the complimentary sequence of CACCTG, we confirmed, using our new method, that CAGG(CC)TG is the Slug-binding motif. Many E-box motifs (CANNTG) were found in the promoter regions of the 20S subunit genes. Among these motifs, only binding sequences with high scores (CACCTG, CAGGTG, CACGTG, and CAGCTG) are shown in Fig. 2. PSMA3, PSMA4, PSMA6, PSMB2, PSMB3, and PSMB5 contained clusters of E-box sequences. When Slug was expressed, PSMB2 and PSMB3 showed a dramatic decrease in their expressions. We did not measured expression of PSMA genes; thus, based on PMSB expression results, dramatic decrease may have occurred, because PSMB2 and PSMB3 have several E-box sequences within the -1 kb region. Proteasome activity is controlled in various ways, such as regulation through the ubiquitination system, interacting proteins, changing subunits, and modification of transcription (Aiken et al.,

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2011). Transcriptional control of house-keeping proteasome genes was first described in the yeast S. cerevisiae. Mannhaupt and colleagues (1999) noted the presence of a common sequence element (GGTGGCAAA) termed proteasome-associated control element (PACE) in the promoters of nearly all genes encoding proteasome subunits. Rpn4, a 60-kDa protein containing a C2H2type zinc finger motif and two acidic domains, was identified as the transcriptional activator that binds to PACE sequences (Mannhaupt et al., 1999). However, the transcriptional regulation of the proteasome in higher eukaryotes has been reported in few studies. Up-regulation of proteasome expression has also been observed in mammalian cells as an adaptive cellular response (Ding et al., 2003; Klimaschewski et al., 2006; Pickering et al., 2010). In the immunoproteasome, alternative catalytic β subunits, β1i, β2i, and β5i, are incorporated upon induction of interferon-γ expression (Griffin et al., 1998). The transcription factor 11 (long isoform of Nrf1) and NF-E2-related factor 2 (Nrf2) were shown to promote the expression of several proteasome genes, and two antioxidant responsive elements (AREs) and were identified in the promoter region of proteasome α subunit 3 (PSMA3) (Kwak et al., 2003; Takabe et al., 2006). In Nrf1-deletion mice, loss of Nrf1 led to impaired proteasome function and neurodegeneration. Gene expression profiling and RT-PCR analysis revealed a coordinated down-regulation of various proteasomal genes, including PSMB6, which encodes a catalytic subunit of the proteasome (Lee et al., 2011). Xu et al. (2012) reported that the CCAAT box-binding transcription factor NF-Y regulates the basal expression of proteasome genes. In the present study, we showed that the EMT-related transcription factor Slug/Snail2 can repress transcription of proteasome subunit genes. Because E-box sequence can be recognized by various EMT-related transcription factors such as Snail1 and Twist, it is hard to say that Slug is the only transcription factor involved in the repression or down-regulation of proteasome activity. Further studies are needed to determine whether the Snail1 and Twist are also involved in the down-regulation of proteasome activity. To the best of our knowledge, this is the first study to investigate the transcriptional modification of proteasome subunit genes that are involved in EMT. Acknowledgment This work was supported by a grant from the National Research Foundation (NRF; grant number 2010-0024-557 to M. Cho), Republic of Korea.

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