Kr-pok increases FASN expression by modulating the DNA binding of SREBP-1c and Sp1 at the proximal promoter
2012; Elsevier BV; Volume: 53; Issue: 4 Linguagem: Inglês
10.1194/jlr.m022178
ISSN1539-7262
AutoresBu‐Nam Jeon, Kim Ys, Won‐Il Choi, Dong‐In Koh, Min-Kyeong Kim, Jae-Hyeon Yoon, Minyoung Kim, Benjamin Hur, Philip Dong-Hyun Paik, Man‐Wook Hur,
Tópico(s)Cholesterol and Lipid Metabolism
ResumoKr-pok (kidney cancer-related POZ domain and Krüppel-like protein) is a new proto-oncogenic POZ-domain transcription factor. Fatty acid synthase gene (FASN) encodes one of the key enzymes in fatty acids synthesis and is the only enzyme that synthesizes fatty acids in cancer cells. Sp1 and SREBP-1c are the two major transcription activators of FASN. We investigated whether Kr-pok modulates transcription of the FASN. FASN expression is significantly decreased in Kr-pok knockout murine embryonic fibroblasts. Coimmunoprecipitation, GST fusion protein pull-down, and immunocytochemistry assays show that the zinc-finger domain of Kr-pok interacts directly with the bZIP DNA binding domain of SREBP-1. Electrophoretic mobility shift assay, oligonucleotide pull-down, and chromatin immunoprecipitation assays showed that Kr-pok changes the transcription factor binding dynamics of Sp1 and SREBP-1c to the SRE/E-box elements of the proximal promoter. We found that Kr-pok expression increased during 3T3-L1 preadipocyte differentiation and that FASN expression is decreased by the knockdown of Kr-pok. Kr-pok facilitates the SREBP-1c-mediated preadipocyte differentiation and/or fatty acid synthesis. Kr-pok may act as an important regulator of fatty acid synthesis and may induce rapid cancer cell proliferation by increasing palmitate synthesis. Kr-pok (kidney cancer-related POZ domain and Krüppel-like protein) is a new proto-oncogenic POZ-domain transcription factor. Fatty acid synthase gene (FASN) encodes one of the key enzymes in fatty acids synthesis and is the only enzyme that synthesizes fatty acids in cancer cells. Sp1 and SREBP-1c are the two major transcription activators of FASN. We investigated whether Kr-pok modulates transcription of the FASN. FASN expression is significantly decreased in Kr-pok knockout murine embryonic fibroblasts. Coimmunoprecipitation, GST fusion protein pull-down, and immunocytochemistry assays show that the zinc-finger domain of Kr-pok interacts directly with the bZIP DNA binding domain of SREBP-1. Electrophoretic mobility shift assay, oligonucleotide pull-down, and chromatin immunoprecipitation assays showed that Kr-pok changes the transcription factor binding dynamics of Sp1 and SREBP-1c to the SRE/E-box elements of the proximal promoter. We found that Kr-pok expression increased during 3T3-L1 preadipocyte differentiation and that FASN expression is decreased by the knockdown of Kr-pok. Kr-pok facilitates the SREBP-1c-mediated preadipocyte differentiation and/or fatty acid synthesis. Kr-pok may act as an important regulator of fatty acid synthesis and may induce rapid cancer cell proliferation by increasing palmitate synthesis. Fatty acid synthase (FASN), a 250- to 270-kDa cytosolic protein and one of the main lipogenic enzymes in mammals, catalyzes reactions that contribute to the conversion of acetyl-CoA and malonyl-CoA to palmitate (1Swinnen J.V. Van Veldhoven P.P. Timmermans L. De Schrijver E. Brusselmans K. Vanderhoydonc F. Van de Sande T. Heemers H. Heyns W. Verhoeven G. Fatty acid synthase drives the synthesis of phospholipids partitioning into detergent-resistant membrane microdomains.Biochem. Biophys. Res. Commun. 2003; 302: 898-903Crossref PubMed Scopus (214) Google Scholar–2Chirala S.S. Wakil S.J. Structure and function of animal fatty acid synthase.Lipids. 2004; 39: 1045-1053Crossref PubMed Scopus (139) Google Scholar, 3Costello L.C. Franklin R.B. 'Why do tumour cells glycolyse?': from glycolysis through citrate to lipogenesis.Mol. Cell. Biochem. 2005; 280: 1-8Crossref PubMed Scopus (132) Google Scholar, 4Menendez J.A. Lupu R. 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Koh D.I. Kim M.H. Kim Y.R. Lee C.E. Kim K.S. et al.Proto-oncogene FBI-1 (Pokemon) and SREBP-1 synergistically activate transcription of fatty-acid synthase gene (FASN).J. Biol. Chem. 2008; 283: 29341-29354Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Kr-pok, a recently characterized proto-oncoprotein, contains a POZ-domain and four Krüppel-like ZFs that are similar to those of FBI-1 in two key functional domains: the POZ-domain (81% similarity) and the four Krüppel-like ZFs (88% similarity) (supplementary Fig. I) (28Lee K.M. Choi W.I. Koh D.I. Kim Y.J. Jeon B.N. Yoon J.H. Lee C.E. Kim S.H. Oh J. Hur M.W. The proto-oncoprotein KR-POK represses transcriptional activation of CDKN1A by MIZ-1 through competitive binding.Oncogene. 2011; (In press)Google Scholar, 29Jeon B.N. Kim M.K. Choi W.I. Koh D.I. Hong S.Y. Kim K.S. Kim M. Yun C.O. Yoon J. Choi K.Y. Hur M.W. et al.KennethKenneth, and M. W. Hur. 2011. KR-POK interacts with p53 and represses CDKN1A transcriptional activation by p53.in: Cancer Res.2011Google Scholar). Accordingly, Kr-pok is expected to have some properties comparable to those of FBI-1, such as adipocyte differentiation and cell proliferation. In this study, we found that Kr-pok and SREBP-1c interact to synergistically activate FASN expression. Kr-pok changes the binding dynamics of SREBP-1c and Sp1 at the core regulatory elements of the FASN promoter, which results in the transcriptional up-regulation of FASN. Kr-pok may be one of the key regulators of fatty acid synthesis and cancer cell proliferation. Stable HEK293T-Rex-Kr-pok cells, which are inducible by doxycycline, were prepared by transfecting mammalian Flp-InTM T-RExTM host HEK293 cells with pOG44 and pcDNA5/FRT/TO©-Kr-pok plasmids and selecting with hygromycin and blasticidin (Invitrogen, Carlsbad, CA). To prepare Kr-pok+/+ and Kr-pok−/− mouse embryonic fibroblasts (MEFs), pregnant female Kr-pok+/− mice were mated with Kr-pok+/− male mice and euthanized at 13.5 days post coitum. The embryos were homogenized, treated with a trypsin-EDTA solution, and placed in a CO2 incubator for 6 h. Fresh DMEM was added to the culture media, and embryo homogenates were continuously incubated to obtain MEFs. HEK293A, HCT116, and LNcaP cells were cultured in DMEM (Gibco-BRL, Gaithersburg, MD) supplemented with 10% FBS (Gibco-BRL). Various fatty acid synthase promoter-luciferase reporter gene plasmids were provided by Dr. Kyung-Sup Kim of the Yonsei University School of Medicine (Seoul, Korea) and Dr. Timothy F. Osborne of the Sanford/Burnham Medical Research Institute (Orlando, FL). pcDNA3.1-Kr-pok-Myc-His and pcDNA3.0-FLAG-Kr-pok plasmids were prepared by cloning mouse brain cDNA fragments into pcDNA3.1 or pcDNA3.0 plasmids (Invitrogen). pcDNA3.1-Myc-SREBP-1c was prepared by cloning pCMV-SREBP-1c into the pcDNA3.1 plasmid (Invitrogen). To prepare recombinant GST-POZ Kr-pok, GST-ZF Kr-pok, and GST-bZIP SREBP-1 proteins, cDNA fragments encoding Kr-pok POZ, Kr-pok zinc fingers, and the SREBP-1 bZIP domain, respectively, were cloned into pGEX4T3 (Amersham Biosciences, Piscataway, NJ). All plasmid constructs were verified by sequencing. Total RNA was isolated from cells using TRIzol reagent (Invitrogen). cDNA was synthesized using 5 μg of total RNA, random hexamers (10 pmol), and superscript reverse transcriptase II (200 units) in a total volume of 20 μl using a reverse transcription kit (Invitrogen). PCR was performed using the following amplification conditions: 94°C denaturation for 3 min, 30 cycles of amplification reaction (94°C for 30 s, 55°C for 30 s, 72°C for 30 s), and a final extension reaction at 72°C for 5 min. Quantitative PCR was performed using the SYBR Green PCR Master Mix (Applied Biosystems, Carlsbad, CA) and ABI PRISM 7300 RT-PCR System (Applied Biosystems). GAPDH mRNA was used as a control. Cells were harvested and lysed in RIPA buffer. Cell extracts (30 µg) were separated using 12% SDS-PAGE, transferred to an Immun-BlotTM PVDF Membrane (Bio-Rad, Hercules, CA), and blocked in 5% skim milk (BD Biosciences, Sparks, MD). Blotted membranes were incubated with antibodies against FLAG-tag (Abcam, Cambridge, UK), GAPDH (Chemicon, Temecula, CA), SREBP-1, Sp1, and Myc-Tag (SantaCruz Biotech, Santa Cruz, CA) and then incubated with HRP-conjugated mouse, rabbit, or goat IgGs (Vector Laboratory, Burlingame, CA). A rabbit polyclonal antibody against Kr-pok by us as reported elsewhere (29Jeon B.N. Kim M.K. Choi W.I. Koh D.I. Hong S.Y. Kim K.S. Kim M. Yun C.O. Yoon J. Choi K.Y. Hur M.W. et al.KennethKenneth, and M. W. Hur. 2011. KR-POK interacts with p53 and represses CDKN1A transcriptional activation by p53.in: Cancer Res.2011Google Scholar). Protein bands were visualized with an ECL solution (PerkinElmer, Waltham, MA). Cells (HCT116, LNcaP) infected with the recombinant adenovirus overexpressing Kr-pok, the shRNA Kr-pok knock-down, and the control adenovirus were cultured for 2 days, harvested, and lysed in RIPA buffer. FASN enzyme activity was analyzed by spectrophotometrically measuring the oxidation of NADPH by the conversion of Malonyl-CoA to palmitate. The reaction was initiated by adding 100 μg of cell lysates to the assay mixture (0.1 M potassium phosphate buffer [pH 7.0], 30 µM Acetyl-CoA, 100 µM Malonyl-CoA, 0.1 mM NADPH, and 1 mM EDTA). The decrease in absorbance at 340 nm was monitored over 30 min. The oxidation of cell lysates was background corrected for NADPH oxidation in the presence of only Malonyl-CoA and Acetyl-CoA. Cells were washed, pelleted, and resuspended in a lysis buffer that was supplemented with protease inhibitors. The cell lysate was precleared, and the supernatant was incubated with antibodies on a rotating platform at 4°C overnight, followed by incubation with protein A-Sepharose Fast Flow beads. The beads were collected, washed, and resuspended in equal volumes of 5× SDS loading buffer. Immunoprecipitated proteins were separated with 12% SDS-PAGE. The Western blot assay was performed as described above. Recombinant GST, GST-POZ Kr-pok, GST-ZF Kr-pok, and GST-bZIP SREBP-1 fusion proteins were prepared from Escherichia coli BL21 (DE3) cells grown overnight at 18°C in medium containing 0.2 mM IPTG. The E. coli were lysed and purified using glutathione-agarose 4 bead affinity chromatography (Peptron, Taejeon, Korea). The purified proteins were then resolved with 12% SDS-PAGE to quantitate and assess purity. Kr-pok and SREBP-1c polypeptides were prepared using the TNT extract in the presence of [35S]methionine (Promega, Madison, WI). GST fusion protein-agarose bead complexes were incubated with in vitro translated [35S]methionine (1175.0 Ci/mol) labeled Kr-pok or SREBP-1c polypeptides at 4°C for 4 h in HEMG buffer. The reaction mixtures were centrifuged, the pellets were rinsed, and the bound proteins were separated using 12% SDS-PAGE. The gels were then exposed to X-ray film (Kodak, Rochester, NY). HEK293A cells were grown on coverslips placed in a culture dish. The cells were then transfected with pcDNA3.0-FLAG-Kr-pok and pcDNA3.1-SREBP-1c-Myc plasmids. After 24 h, the cells were washed with cold PBS and fixed in 97:3 cold methanol:formaldehyde for 20 min at −20°C. The cells were permeabilized in 0.2% Triton X-100 and washed with PBS. Next, the cells were incubated in 5% normal horse serum and then incubated with mouse anti-FLAG primary antibody for 2 h at room temperature. The cells were washed and incubated with FITC-conjugated anti-mouse IgG secondary antibody (Invitrogen). For double staining, the cells were washed and incubated with rabbit anti-Myc antibody and then with Rhodamine-conjugated anti-rabbit IgG secondary antibody (Invitrogen). After DAPI staining, washing, and mounting, the immunostained cells were examined on a Carl Zeiss LSM 510 confocal laser scanning microscope (Carl Zeiss, Germany). HEK293A cells were transiently cotransfected with the SREBP-1c expression plasmid, increasing amounts of the Kr-pok expression plasmid, and the reporter plasmid (pGL2-6xSRE-Luc, pGL2-FAS1, 2, 3-Luc, and pGL3-FASN Wt or Mt-Luc reporter fusion plasmids) using Lipofectamine Plus reagent (Invitrogen). After 24 h of incubation, the cells were harvested and analyzed for luciferase activity. The reporter activity was normalized with cotransfected β-galactosidase activity or protein concentration for transfection efficiency. The oligonucleotide probes were annealed by heating at 93°C for 5 min and then slowly cooled to room temperature. The annealed oligonucleotides were prepared by labeling with [α-32P]ATP and Klenow enzyme by incubating for 30 min at 37°C (Roche, Nutley, NJ). 32P-labeled, double-stranded oligonucleotide probes were purified with SephadexTM G-50 (Amersham Biosciences, Uppsala, Sweden). The sequences of the oligonucleotides used in the EMSA are as follows (only the top strand is listed): FASN SRE/E-box: 5′-GATCGTCCAGCCCATGTGGCGTGGC-3′. The remaining procedure was performed as reported previously (27Choi W.I. Jeon B.N. Park H. Yoo J.Y. Kim Y.S. Koh D.I. Kim M.H. Kim Y.R. Lee C.E. Kim K.S. et al.Proto-oncogene FBI-1 (Pokemon) and SREBP-1 synergistically activate transcription of fatty-acid synthase gene (FASN).J. Biol. Chem. 2008; 283: 29341-29354Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). HEK293A cells transfected with pcDNA3.0-FLAG-Kr-pok and/or pcDNA3.0-SREBP-1c-Myc were lysed in HKMG buffer (10 mM HEPES [pH 7.9], 10 mM KCl, 5 mM MgCl2, 10% glycerol, 1 mM dithiothreitol, and 0.5% Nonidet P-40). The cellular extracts were incubated with 1 μg of biotinylated double-stranded oligonucleotides. The sequence of the SRE/E-box oligonucleotide was 5′-GTCCAGCCCATGTGGCGTGGC-3′ (only the top strand is listed). The mixtures were incubated with Streptavidin-agarose beads for 2 h to collect the DNA-protein complex and then spun, and the pellets were washed with HKMG buffer. The precipitates were resolved by 10% SDS-PAGE and analyzed using Western blot. Using chromatin immunoprecipitation (ChIP) assays, we investigated the molecular interaction between Sp1 or SREBP-1c and the SREBP-1c binding site on the FASN promoter in the presence of Kr-pok. Subconfluent HEK293A cells on a culture dish were transfected with pcDNA3.0-FLAG Kr-pok and/or pcDNA3.1-SREBP-1c using Lipofectamine Plus and grown for an additional 48 h. The cells were fixed with formaldehyde (final concentration, 1%) to cross-link Kr-pok, SREBP-1c, and Sp1 onto the FASN promoter. The remaining ChIP procedures were performed as reported previously (27Choi W.I. Jeon B.N. Park H. Yoo J.Y. Kim Y.S. Koh D.I. Kim M.H. Kim Y.R. Lee C.E. Kim K.S. et al.Proto-oncogene FBI-1 (Pokemon) and SREBP-1 synergistically activate transcription of fatty-acid synthase gene (FASN).J. Biol. Chem. 2008; 283: 29341-29354Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). PCR reactions of immunoprecipitated DNA were carried out using oligonucleotide primer sets designed to amplify the FASN gene region. FASN WT or MT primers (forward: 5′-CAGGCGCGTTCCCGCGCAGG-3′; reverse: 5′-GAGAGCGAGGCTGGAGCGCG-3′) and FAS2 or FAS3 primers (forward: 5′-TCCAAACTCATCAATGTA-3′; reverse: 5′-AAAGCAATTGTTCCAGGAACCAGGG-3′) were used for the ChIP assays. The Kr-pok cDNA was cloned into an adenovirus E1 shuttle vector pCA14 (Microbix, Mississauga, Ontario, Canada) to generate pCA14-Kr-pok. To prepare a recombinant adenovirus expressing shRNA against Kr-pok, annealed shRNA DNA sequences (sense: 5′-GATCCCTCCAGTGCATCGTGAATGTTTTTCAAGAGA(loop)-ACATTCACGATGCACTGGATTTTTTTGGAA(loop)-A-3′; antisense: 5′-AGCTTTTCCAAAAA(loop)-AATCCAGTGCATCGTGAATGTTCTCTTGAA(loop)-AAACATTCACGATGCACTGGAGG-3′) were cloned into pSilencer 2.0-U6 (Ambion, Austin, TX) and subcloned into the pΔE1sp1A vector. The pCA14-Kr-pok shuttle vector or pΔE1sp1A-U6-shKr-pok vector and the adenovirus vector vmdl324Bst were linearized by restriction enzyme digestion for homologous recombination into E. coli BJ518. The homologous recombinant adenoviral plasmid was digested and transfected into HEK293 cells to generate the adenovirus expressing Kr-pok (dl324-Kr-pok) or shRNA against Kr-pok (dl324-shKr-pok). siRNAs against Srebp-1c mRNA were designed and purchased from Bioneer (Seoul, Korea): siSrebp-1c, 5′-CCACGGAGCCAUGGAUUGCACAUUUdTdT-3′, 5′-AAAUGUGCAAUCCAUGGCUCCGUGGdTdT-3′. The siRNAs (50 pmol) were transfected into 3T3-L1 cells using LipofectamineTM RNAiMAX (Invitrogen). 3T3-L1 preadipocytes were maintained at low passage and grown to confluence in DMEM supplemented with 10% calf serum (Gibco-BRL). Differentiation was induced by placing 2 day postconfluent cultures in DMEM supplemented with 10% calf serum for up to 8 days. The medium for differentiating 3T3-L1 preadipocytes was supplemented with 0.525 mM methylisobutylxanthine (Sigma, MO), 1 μM dexamethasone (Sigma), and 0.167 μM insulin (Roche). Forty-eight hours later, this medium was replaced with medium supplemented only with 0.167 μM insulin (Roche). Kr-pok, recently characterized as a proto-oncoprotein expressed abundantly in most kidney cells, is similar to FBI-1 at the amino acid sequence level in two key functional domains (Supplementary Fig. I) (28Lee K.M. Choi W.I. Koh D.I. Kim Y.J. Jeon B.N. Yoon J.H. Lee C.E. Kim S.H. Oh J. Hur M.W. The proto-oncoprotein KR-POK represses transcriptional activation of CDKN1A by MIZ-1 through competitive binding.Oncogene. 2011; (In press)Google Scholar, 29Jeon B.N. Kim M.K. Choi W.I. Koh D.I. Hong S.Y. Kim K.S. Kim M. Yun C.O. Yoon J. Choi K.Y. Hur M.W. et al.KennethKenneth, and M. W. Hur. 2011. KR-POK interacts with p53 and represses CDKN1A transcriptional activation by p53.in: Cancer Res.2011Google Scholar). FBI-1 expression has been shown to increase during the 6- to 48-h time period of human preadipocyte differentiation, and it was suggested that FBI-1 may play an important role in adipogenesis and rapid cancer cell growth (26Laudes M. Christodoulides C. Sewter C. Rochford J.J. Considine R.V. Sethi J.K. Vidal-Puig A. O'Rahilly S. Role of the POZ zinc finger transcription factor FBI-1 in human and murine adipogenesis.J. Biol. Chem. 2004; 279: 11711-11718Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 27Choi W.I. Jeon B.N. Park H. Yoo J.Y. Kim Y.S. Koh D.I. Kim M.H. Kim Y.R. Lee C.E. Kim K.S. et al.Proto-oncogene FBI-1 (Pokemon) and SREBP-1 synergistically activate transcription of fatty-acid synthase gene (FASN).J. Biol. Chem. 2008; 283: 29341-29354Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). In this investigation, we tested whether Kr-pok regulates FASN gene expression. RT-PCR of mRNAs and Western blot analysis of total cell lysates prepared from MEF cells revealed that the knockout of the Kr-pok gene decreased FASN gene expression (Fig. 1A, B). Kr-pok induction of stable HEK293T-REx-Kr-pok cells by doxycyclin increased FASN gene expression (Fig. 1C, D). We investigated whether Kr-pok can regulate transcription of FASN gene in cancer cells. RT-qPCR and Western blot analysis of the cell lysates prepared from HCT116 (colon cancer) and LNcaP (prostate cancer) cells infected with recombinant dl324-Kr-pok adenovirus revealed that Kr-pok increases transcription of the FASN gene and FASN expression in cancer cells (Fig. 1E, F, H, I). We also analyzed lipogenic activity at the enzyme level by monitoring the oxidation of NADPH to NADP+ caused by the conversion of Malonyl-CoA and Acetyl-CoA to palmitate. Whereas ectopic Kr-pok increased FASN enzyme activity (ΔC/mg protein/min.; ΔC =ΔA/E; ΔA = change in absorbance; E = extinction coefficient of NADPH [E340nm= 6.22 mM−1 cm−1]), the knock-down of Kr-pok expression clearly decreased FASN enzyme activity in the two cancer cell lysates tested (Fig. 1G, J). These data show that the transcription of the FASN gene, a major player in the synthesis of the phospholipids of the cell membrane, was potently activated by Kr-pok. SREBPs are major transcription regulators that control the expression of enzym
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