Biochimica et Biophysica Acta 1839 (2014) 288–296

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Activating transcription factor 4 mediates up-regulation of alanine aminotransferase 2 gene expression under metabolic stress María C. Salgado, Isidoro Metón, Ida G. Anemaet 1, Isabel V. Baanante ⁎ Departament de Bioquímica i Biologia Molecular, Facultat de Farmàcia, Universitat de Barcelona, Joan XXIII s/n, 08028 Barcelona, Spain

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Article history: Received 22 October 2013 Received in revised form 18 December 2013 Accepted 6 January 2014 Available online 10 January 2014 Keywords: Alanine aminotransferase 2 Alanine aminotransferase 1 Activating transcription factor 4 Amino acid deprivation ER stress Gene knock-down

a b s t r a c t Alanine aminotransferase (ALT) provides a molecular link between carbohydrate and amino acid metabolism. In humans, two ALT isoforms have been characterized: ALT1, cytosolic, and ALT2, mitochondrial. To gain insight into the transcriptional regulation of the ALT2 gene, we cloned and characterized the human ALT2 promoter. 5′-deletion analysis of ALT2 promoter in transiently transfected HepG2 cells and sitedirected mutagenesis allowed us to identify ATF4 as a new factor involved in the transcriptional regulation of ALT2 expression. Quantitative RT-PCR assays showed that the metabolic stressors histidinol and tunicamycin increased ATF4 levels and up-regulated ALT2 in HepG2 and Huh7 cells. Consistently, knockdown of ATF4 decreased ALT2 mRNA levels in HepG2 and Huh-7 cells. Moreover, ATF4 silencing prevented the activating effect of histidinol and tunicamycin on ATF4 and ALT2 expression. Our findings point to ALT2 as an enzyme involved in the metabolic adaptation of the cell to stress. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Alanine aminotransferase (ALT; EC 2.6.1.2) links carbohydrate and amino acid metabolism through catalysing the reversible transamination between L-alanine and 2-oxoglutarate to form pyruvate and L-glutamate. In mammals, two ALT isoforms, ALT1, cytosolic, and ALT2, mitochondrial, each encoded by a different gene, have been identified [1–4]. Western blotting indicates human expression of ALT1 in the liver, skeletal muscle, kidney and to a lesser extent, in the heart, whereas ALT2 is mainly expressed in the heart and skeletal muscle [5]. High levels of ALT activity are associated with hepatic injury conditions such as hepatitis, cirrhosis, and drug hepatotoxicity [6–8], and to other pathologies such as obesity, muscle diseases, type 1 diabetes, coronary atherosclerotic disease, and metabolic syndrome [9–17]. In addition, a rise of serum ALT activity within the reference values in healthy populations is considered a promising biomarker for the development of insulin resistance and type 2 diabetes [11,18–22]. Recent research efforts were focused to develop better tools for the diagnosis and prognosis of the various diseases associated with increased ALT activity. However, little attention has been paid to unravel specific mechanisms that govern expressional regulation of cytosolic and mitochondrial ALT. Gray et al. [23] reported reduction of ALT1 mRNA levels, but not ALT2 mRNA levels, in the liver of Krüppel-like

⁎ Corresponding author. Tel.: +34 934024521; fax: +34 934024520. E-mail address: [email protected] (I.V. Baanante). 1 Present address: Section Molecular and Developmental Genetics, Institute of Biology, Leiden University, Sylvius Laboratory, Sylviusweg 72, 2333 BE Leiden, The Netherlands. 1874-9399/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbagrm.2014.01.005

factor 15-deficient mice. Takashima et al. [24] showed that KLF15 silencing significantly reduced hepatic ALT1 mRNA levels in mice. Transfection experiments in Huh-7 cells showed ALT1 promoter induction by the peroxisome proliferator-activated receptors (PPARs) α and γ together with RXRα, while the human ALT2 promoter was unaffected [25]. Recently, Josekutty et al. [26] showed that phospho-c-Jun binds and transactivates ALT1 and AST1 promoters but not ALT2 or AST2 promoters in the presence of a microsomal triglyceride transfer protein (MTP) inhibitor in Huh-7 cells. In Sparus aurata, Anemaet et al. [27] showed transactivation of the cALT promoter by p300 and cMyb and that p300 is involved in the up-regulation of cALT2 mRNA levels in the liver of streptozotocin (STZ)-treated S. aurata. In this species, Salgado et al. [28] demonstrated that HNF4α regulates mALT gene transcription in the kidney of starved- and STZ-treated fish. Activating transcription factor 4 (ATF4) is a member of the basic region leucine zipper (bZIP) transcription factor family; it can regulate gene transcription by forming a homodimer or heterodimer with other bZIP transcription factors [29]. ATF4 can bind to promoter regions of target genes containing the following conserved sequences: the cAMP response element (CRE) site (sequence: 5′-GTGACGTACAG-3′), and C/EBP-ATF response elements (CARE) (sequence: 5′-TGATGXAAX-3′) [30,31]. ATF4 is a stress responsive gene, which is up-regulated under stressful conditions, including oxygen deprivation (hypoxia/anoxia), amino acid deprivation, endoplasmic reticulum stress (ER stress), oxidative stress, and by the growth factor heregulin [32–35]. The various stress signals integrate to a common pathway of increased translation of ATF4, which subsequently ensures supply of amino acids for protein biosynthesis and protects cells against oxidative stress, by modulating a number of genes involved in mitochondrial function (e.g., Lon

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mitochondrial protease homologue), amino acid metabolism and transport (e.g. asparagine synthetase), as well as in redox chemistry (e.g., NADH-cytochrome B5 reductase homolog) [32,36]. Besides playing a role in amino acid and glucose deprivation, recent reports also support involvement of ATF4 in lipid metabolism and in regulation of energy homeostasis [31,37–39]. Since molecular studies devoted to unravel regulation of ALT expression in mammals are scarce, the aim of this study was to characterize the molecular control of ALT transcription to increase the current knowledge about the role exerted by ALT isoenzymes in the cell. To this end, we addressed the role of ATF4 in the transcriptional control of ALT1 and ALT2 in HepG2 and Huh-7 cells under stressful conditions.

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tobacco acid pyrophosphatase and a 5′ RACE Adapter oligonucleotide from the kit was ligated to 5′ non-dephosphorylated RNAs to perform a reverse transcription reaction. A nested PCR to amplify the 5′ end of ALT2 mRNA was performed following the manufacturer's instructions and gene-specific primers MC10-1 (primary PCR; Table 1) and MC10-1N (secondary PCR; Table 1). The single 198 bp band generated was purified and ligated into a pGEM-T Easy plasmid (Promega). Identical nucleotide sequence corresponding to the 5′ end of ALT2 cDNA was obtained by sequence analysis of three independent clones.

2.3. Construction of reporter gene plasmids 2. Materials and methods 2.1. Cloning of the 5′-flanking region of ALT2 and ALT1 human genes The 5′-flanking region of human ALT1 and ALT2 was isolated by PCR. To accomplish this, gene-specific primers IG05/IG03 for ALT1 and MC0497/MC0505 for ALT2 (Table 1) were used together with human genomic DNA (Roche) as a template. The resulting 2139 bp (ALT1) and 2004 bp (ALT2) amplicons were ligated into the pGEM-T Easy plasmid (Promega) to generate pGEMALT1 − 2139 and pGEMALT2 − 2004. Two independent clones were fully sequenced on both strands according to the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit instructions (Applied Biosystems). 2.2. Characterisation of ALT2 transcription start site The 5′ end of the human ALT2 mRNA was determined using the FirstChoice® RLM-RACE Kit (Ambion). This approach amplifies cDNA only from full-length, capped mRNA. To this end 10 μg of total RNA obtained from HepG2 cells was treated with calf intestine alkaline phosphatase (CIP) in order to remove free 5′-phosphates from nucleic acids, such as fragmented mRNA, without affecting the cap structure found on intact 5′ ends of mRNA. Thereafter, the cap structure was removed with Table 1 Primers used in the present study. Primer

Sequence (5′ to 3′)

IG05 IG03 HAC01 HAC03 MC0505 MC0497 MC06H1 MC06H2 MC06H5OK MC0950

GTGGGGAGTGGGTTGTGCTGTTTGG GACTCTACCCAGACCAGGCGGGAAGG GAGCTAGCGAGTGGGTTGTGCTGTTTGG GCAAGCTTGACTCTACCCAGACCAGGCG GCGCGCTTGCGGAGAGAAACCCTG GGGTGGACAGCAAGGGACTGCATG GGACGCGTGGACATTACATGC GGAAGCTTGCGGAGAGAAACCCTGGCAC GGACGCGTCGAAGCCAGGCTGGCACCGC GACGCGTGTGGCCAAGTCCCAGACTCCTCACCCGCCTTCCTGCCAGCTC TGGTGGGCAGCCGCAGGCACCGTGTGGCCTTGGACCCTGAAACTCGGG GCGATGACTGC GAAGCTTGCCGCCATGACCGAAATGAGCTTCCTG GGGATCCCTAGGGGACCCTTTTCTTCC CGACCAGTCGGGTTTGGGGGC AGCCCGCCTTAGCCTTGTCGC CGGCTACCACATCCAAGGAA GCTGGAATTACCGCGGCT CATGAACCCGCAGGTGAAGG TGACCTCTGTGAATGGCTTTTTG GAGCACAGGTGACCGGAGCCA CCTGCTCCAGCTCCAAGGCTCGCT GTTCATGGACTCCAGCGTGAGGATG GGAGCTGGGGGTCCGGGGACCACAG CATTGTAGCCCTCTGTGTGCTC CCAGCAGGTCAGCAAAGAATTT

MC0955 MC0956 MC104 MC105 MC109 MC110 MC0916 MC0917 MC100 MC101 MC10-1 MC10-1N JDHPRTrt1S JDHPRTrt1AS

The following primers contain restriction sites indicated in bold and underlined: HAC01, NheI; MC06H2, MC0955 and HAC03, HindIII; MC0956 BamHI; MC06H1, MC06H5OK and MC0950, MluI.

The human ALT1-luciferase reporter fusion construct pGALT1 −1911, which harbours promoter sequences located between positions −1911 and +223 relative to the transcription start site [25], was generated by PCR. To this end, the gene-specific primers HAC01 and HAC03 (Table 1) were used together with pGEMALT1 − 2139 as a template. The PCR product was restricted with NheI/HindIII and ligated into the pGL3-Basic promoterless luciferase reporter plasmid (Promega) previously digested with the same enzymes. The human ALT2-luciferase reporter fusion construct (pGALT2 −1679; −1679 and +325 relative to the transcription start site) was generated by PCR using the gene-specific primers MC06H1 and MC06H2 (Table 1) together with pGEMALT2 − 2004 as a template. Following restriction with MluI/HindIII, the PCR product was ligated into the pGL3-Basic promoterless luciferase reporter plasmid (Promega) previously digested with the same enzymes. To obtain the pGALT2 − 1534 reporter construct (−1534 to +325 bp), pGALT2 −1679 was digested with MluI and MlsI, the digested plasmid was filled and dephosphorylated by treatment with CIP (Sigma) for 1 h and was thereafter ligated to an MlsI/MlsI fragment (corresponding to nucleotides −1534 to −498) obtained from digestion of pGALT2 − 1679 with MlsI. Reporter construct pGALT2 − 497 (−497 to +325 bp) was obtained by self-ligation of filled-in ends of pGALT2 − 1679 after digestion with MlsI/MluI. Construct pGALT2 − 172 (−172 to +325 bp) was obtained by digesting pGALT2 − 1679 with RsrII/MluI followed by chew-back and filled-in reactions with Klenow enzyme and self-ligation. The pGALT2 − 90 (−90 to +325 bp) construct was obtained by digesting pGALT2 − 1679 with ApaI/MluI followed by chew-back and filled-in reactions with Klenow enzyme and selfligation. The pGALT2 + 195 (+195 to +325 bp) plasmid was obtained by PCR using primer pairs MC06H5OK and MC06H2 (Table 1), and pGALT2 −1679 as a template. The PCR product was digested with MluI and HindIII and ligated to the pGL3-Basic vector, previously digested with the same enzymes. Construct pGALT2 − 1534mut (−1534 to +325 bp), which harbours a mutated ATF4 binding site, was obtained by PCR using oligonucleotides MC0950 and MC06H2 (Table 1). The PCR product was digested with MluI and HindIII and ligated to pGL3-Basic digested with the same enzymes. Then the construct was digested with MlsI, dephosphorylated and ligated to the previously described MlsI fragment (−1534 to −497). All constructs were verified by cycle sequencing.

2.4. Molecular cloning of human ATF4 cDNA RT-PCR was carried out with total RNA from HepG2 cells. The synthesized cDNA was used as a template for PCR using primers MC0955 and MC0956 (Table 1), designed from data bank sequence for human ATF4 (GenBank: D90209.1). The PCR was conducted through 35 cycles of denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s, and DNA synthesis at 72 °C for 2 min, followed by a final extension step of 5 min. The resulting product was restricted with BamHI and HindIII and ligated into pcDNA3, previously digested with the same enzymes, to generate pcDNA3-ATF4. Two independent clones were fully sequenced on both strands.

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2.5. Cell culture, transfection and luciferase assay The human hepatoma derived cell line HepG2 (ATCC HB 8065) was cultured in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% foetal bovine serum, 90 IU/l penicillin, 90 μg/ml streptomycin and 2 mM glutamine. The cells were grown in six-well plates at 37 °C in 5% CO2. The calcium phosphate coprecipitation method was used for the transient transfection of HepG2 cells at 45–55% confluence. Cells were transfected with 2 μg of reporter construct, and when necessary, with 200 ng of expression vector encoding different transcription factors. To correct for variations in transfection efficiency, 250 ng of CMV-β (lacZ) plasmid was included in each transfection. The total amount of DNA added to the cells was kept constant at 2.65 μg by the addition of carrier DNA. The cells were harvested 16 h later, washed in phosphate-buffered saline (PBS) and incubated for 15 min with 300 μl of Cell Culture Lysis Reagent (Promega). Cell debris was removed by centrifugation at 9000 ×g for 30 s, and luciferase activity was measured in the supernatant after the addition of the Luciferase Assay Reagent (Promega) in a TD-20/20 Luminometer (Turner Designs). The activity of β-galactosidase was measured in 20–50 μl of the clear lysate as previously described [40]. Huh-7 hepatocyte derived cellular carcinoma cell line was cultured in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% foetal bovine serum, 90 IU/l penicillin, 90 μg/ml streptomycin and 2 mM glutamine. The cells were grown in six-well plates at 37 °C in 5% CO2.

sample was normalized with human ribosomal-18 s using primer pair MC109/MC110 (Table 1). Variations in gene expression were calculated by the standard ΔΔCt method. 2.9. Western blot analysis Total or nuclear protein extracts from HepG2 cells were separated on 12% SDS–PAGE gels, transferred to Hybond ECL membranes (GE Healthcare Life Sciences) and immunoblotted with anti-ATF4 or ALT2 antibodies (sc-22800 and sc-66914; Santa Cruz Biotechnology) following the ECL Western blotting (Bio-Rad) procedure. An anti-rabbit peroxidase-conjugated secondary antibody was used for chemiluminescent detection (sc-2004; Santa Cruz Biotechnology). Membranes where stripped and immunoblotted with anti-actin antibody (A 2066; Sigma-Aldrich). 2.10. Statistics Data were subjected to one-way and two-way ANOVAs using a computer program (PASW statistics, Hong Kong). One-way ANOVA statistical differences among three or more levels were determined with the Bonferroni post hoc test. One-way statistical analysis with two levels was determined using Student's t-test. 3. Results 3.1. Cloning of the 5′-flanking region of the human ALT2 gene

2.6. Subcellular fractionation of HepG2 cells HepG2 cells were lysed at 4 °C with a Dounce homogeniser in buffer A (50 mM Tris–HCl pH 7.5, 0.5 mM EDTA, 50 mM NaF, 0.5 mM PMSF, 1 mM DTT, 200 mM mannitol, 70 mM sucrose). The homogenate was centrifuged at 500 ×g for 10 min to remove cell debris. The mitochondrial fraction was pelleted by centrifugation at 10,000 ×g for 15 min. The resulting supernatant contained the cytosolic fraction. Mitochondrial pellets were washed twice with buffer A, centrifuged in the same conditions and lysed after resuspension in lysis buffer (50 mM Tris– HCl pH 7.5, 0.5 mM EDTA, 50 mM NaF, 0.5 mM PMSF, 1 mM DTT). ALT activity assayed in the direction of L-glutamate formation and protein content in cytosolic and mitochondrial fractions were measured at 30 °C as described elsewhere [41]. 2.7. siRNA transfection siRNA targeting ATF4 (AM16704) and control siRNA (AM4611) were obtained from Ambion. HepG2 and Huh-7 cell lines were seeded at 2 × 105 cells per well in 6-well plates and transfected with 25 nM of siRNA using X-tremeGENE siRNA Transfection Reagent (Roche) according to the manufacturer's instructions. Forty-eight hours after transfection, 2 mM histidinol or 5 μM tunicamycin were added to the corresponding wells. Six hours after treatment the cells were harvested. 2.8. Quantitative real time PCR The reverse transcriptase reaction was performed on 2 μg of total RNA isolated from cultured HepG2 and Huh-7 cells by incubation with Moloney Murine Leukemia Virus (M-MLV) RT (Invitrogen) for 60 min at 37 °C in the presence of random hexamer primers. The cDNA product was used for subsequent quantitative real time PCR (qRT-PCR) analysis. ALT2, ALT1 and ATF4 mRNA levels were determined in an ABI Prism 7000 Sequence Detection System (Applied Biosystems) using 0.4 μM of each primer (MC0916/MC0917, MC100/MC101 and MC104/MC105 for ALT2, ALT1 and ATF4 respectively; Table 1), 10 μl of SYBR Green (Applied Biosystems) and 1.6 μl of the diluted cDNA mixture in a final volume of 20 μl. The amount of mRNA for the gene of interest in each

A 2004 bp of the 5′-flanking region (−1679 to +325 bp relative to the transcription start site) of the ALT2 gene was isolated by PCR, using oligonucleotides designed from the human genomic sequence. The transcription start site of ALT2 mRNA was determined using the RLMRACE approach [42]. A single 198 bp fragment was obtained and cloned into pGEM-T Easy. Upon sequencing, analysis of three independent clones indicated that human ALT2 mRNA initiates 223 nucleotides upstream from the translation start codon. Sequence analysis of the isolated fragment using the MatInspector program [43], revealed the presence of several putative transcription factor binding sites in the proximal promoter region (Fig. 1A). 3.2. Identification of a functional human ALT2 promoter To determine whether the 5′-flanking region of the ALT2 gene contains a functional promoter, a fragment of the isolated 2004 bp promoter sequence was subcloned into the promoterless plasmid pGL3-Basic, upstream of the firefly luciferase reporter gene. HepG2 cells were transiently cotransfected with the resulting construct (pGALT2 − 1534) and a lacZ-containing plasmid as an internal control for transfection efficiency. The cell lysate was assayed for luciferase and β-galactosidase activity 16 h after transfection. The pGALT2 − 1534 construct exhibited a 146-fold increase in luciferase activity relative to the promoterless vector, pGL3-Basic (Fig. 1B). This result indicates that the region comprised within 1534 nucleotides upstream from the transcription start site of human ALT2 contains a functional promoter. To further examine the functional regions involved in the modulation of basal ALT2 expression in humans, a sequential 5′-deletion analysis of the promoter fragment was performed. Deleted fragments with 5′ ends ranging from −497 to +195 and 3′ ends at +325, were fused to the luciferase reporter gene and transfected into HepG2 cells. Induction of ALT promoter activity in cells transfected with pGALT2 − 497, pGALT2 − 172, pGALT2 − 90 and pGALT2 + 195 resulted in an upregulation of 133-, 55-, 13- and 4-fold, respectively, compared to the empty vector (Fig. 1B). These findings indicate the presence of cisacting elements within 497 bp upstream from the transcriptional start of the human ALT2 gene. The fact that the promoter sequence of human ALT2 has a putative C/EBP-ATF response element (CARE), and

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291

A

B

Fig. 1. 5′ flanking region of ALT2 gene and promoter deletion analysis of the 5′-flanking region of human ALT2 gene in HepG2 cells. (A) Sequence analysis of the 5′-flanking region of the human ALT2 gene. The DNA sequence isolated by PCR is shown in capitals. The arrow indicates the transcription start site. The translation start codon ATG is in boldface and underlined. Several putative binding sites for transcription factors are boxed. (B) The top left part represents genomic organization of the 5′-flanking region of human ALT2 gene. Relevant restriction sites are indicated. Nucleotide numbering starts with +1, corresponding to the transcription initiation site. The human ALT2 promoter fragments having varying 5′ ends and an identical 3′ end (+325) were fused to the luciferase reporter gene in pGL3-Basic vector. The constructs were transfected into HepG2 cells along with CMV-β to normalize for transfection efficiency. Luciferase activity is expressed as a fold increase over promoterless reporter plasmid pGL3-Basic. Results presented are the mean ± SD from six independent experiments performed in duplicate.

that ATF4 is involved in transcriptional regulation of several amino acid metabolism regulatory enzymes [37,44,45], led us to investigate the effect of ATF4 on human ALT2 promoter activity. 3.3. ATF4 transactivates the human ALT2 promoter In silico analysis indicated the presence of a putative ATF4 response element in the human ALT2 promoter at nucleotide position − 425 to − 417 upstream from the transcription start site (Fig. 1A). To analyse the effect of ATF4 on the transcriptional activity of the ALT2 gene, transient transfection experiments were performed in HepG2 cells. Reporter constructs containing sequential 5′-deletions of pGALT2 − 1534 were introduced into HepG2 cells together with an expression plasmid encoding human ATF4. Cotransfection of ATF4

with reporter constructs equal to or longer than pGALT2 − 497 resulted in a 4-fold induction of the transcriptional activity relative to the basal activity of the corresponding promoter construct. These constructs contain an ATF4 response element at position −425 to −417, from the transcription start site. No significant enhancement of promoter activity was observed in the cells transfected with reporter constructs that lack the − 425 to − 417 element (pGALT2 − 172, pGALT2 − 90, pGALT2 + 195 or the promoterless vector pGL3-Basic) (Fig. 2). To study whether the transcriptional action of ATF4 on ALT genes is restricted to ALT2 or it may transactivate also ALT1, a reporter construct carrying ALT1 promoter (pGALT1 − 1911) was transfected into HepG2 cells together with an expression plasmid encoding ATF4. No enhancement of the transcriptional activity of ALT1 was observed (Fig. 2).

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expression plasmids alone did not cause a marked effect on the promoter activity of the different constructs. However, when cotransfected together with the ATF4 expression plasmid, ATF3 and CHOP abolished the transactivation promoted by ATF4 in the reporter constructs containing the CARE elements, pGALT2 − 1534 and pGALT2 − 497 (Fig. 3).

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ATF3 and CHOP were described as negative regulators of ATF4 action in several reports [31]. To address whether ATF3 and CHOP modulate ATF4-dependent transactivation of the ALT2 promoter, we transfected HepG2 cells with pGALT2 − 1534, pGALT2 − 497, pGALT2 − 172, pGALT2 − 90, pGALT2 + 195, pGALT2 − 1534mut or pGL3-Basic together with ATF3 or CHOP expression plasmids alone or in combination with a plasmid encoding ATF4. Transfection with ATF3 or CHOP

ATF4 ATF3 CHOP10 ATF4+ATF3 ATF4+CHOP10

* * *

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3.5. ATF3 and CHOP are transcriptional repressors of ATF4-induced ALT2 promoter activity

* * *

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52 4

A three point mutation in the CARE element located at position − 425 to − 417 of the promoter construct pGALT2 − 1534 was introduced to generate a reporter construct encompassing a mutated ATF4 response element (pGALT2 − 1534mut). Cotransfection experiments with the mutated construct and an expression vector encoding human ATF4 were performed in HepG2 cells to assess whether this transcription factor still remained able to transactivate the mutant reporter construct. As shown in Fig. 2, introduction of three point mutations in the CARE element prevented enhancement of transcriptional activity by human ATF4 in transient transfection experiments. These results further demonstrated that the ATF4 response element, found at position − 425 to − 417 upstream of the transcriptional start of the human ALT2 promoter, is responsible for ALT2 gene transactivation by ATF4. To confirm that ATF4-dependent transactivation of ALT2 results in a rise of ALT2 mRNA and a concomitant increase of mitochondrial ALT activity, HepG2 cells were transiently transfected with a plasmid encoding ATF4 or empty vector. ALT activity in cytosolic and mitochondrial cell extracts and ALT2 mRNA levels were determined 16 h following transfection. Overexpression of ATF4 significantly increased 2.5-fold ALT2 mRNA abundance (normalized ALT2 mRNA levels expressed as mean ± SD: 1.0 ± 0.1 in control cells vs. 2.5 ± 0.3 in ATF4transfected cells; P = 0.019) and 4.1-fold mitochondrial ALT activity (U/mg of ALT activity expressed as mean ± SD: 19.6 ± 6.8 in control cells vs. 81.5 ± 17.9 in ATF4-transfected cells; P = 0.044) while cytosolic ALT activity remained unaffected (U/mg of ALT activity expressed as mean ± SD: 19.6 ± 1.8 in control cells vs. 18.7 ± 2.7 in ATF4transfected cells; P = 0.734).

-1

3.4. Mutating the ATF4 response element abolishes the ATF4 dependent transactivation of human ALT2 gene

Fold induction

1-

AL T

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Fig. 2. ATF4 transactivates human ALT2 promoter in HepG2 cells. HepG2 cells were transfected with promoter reporter constructs for ALT2 (pGALT2 − 1534, pGALT2 − 497, pGALT2 − 172, pGALT2 − 90, pGALT2 + 195 and pGALT2 − 1534mut), ALT1 (pGALT1 − 1911) or the promoterless vector pGL3-Basic, with or without an expression plasmid encoding human ATF4. The promoter activity displayed by the constructs alone was set at 1. Results presented are means ± SD from at least two independent duplicate experiments.

Having concluded that ATF4 binds to and transactivates the human ALT2 promoter whereas the human ALT1 promoter remained unaffected, we studied the role of ATF4 on the transcriptional expression of ALT2 and ALT1 in HepG2 cells under stressful conditions that have previously been reported to up-regulate ATF4 protein levels [31]. First, we tested whether expression of ALT1 and ALT2 is influenced by amino acid deprivation. We therefore made use of L-histidinol, a histidine analogue, which inhibits activation of histidine by histidyl-tRNA synthetase [46]. Analysis by qRT-PCR revealed that treatment with 2 mM histidinol increased ALT2 and ATF4 mRNA levels in HepG2 cells. Between 3 h and 15 h after histidinol treatment, ATF4 and ALT2 levels followed a similar pattern, rising from 2-fold at 3 h to 4-fold at 15 h (Fig. 4A). ATF4 and ALT2 protein levels also increased, more than 5fold and 2-fold, respectively, when measured 6 h after 2 mM histidinol treatment (Fig. 4B). Regarding ALT1, mRNA levels remained similar during the first 8 h following histidinol treatment. Contrary to ALT2, ALT1 expression significantly decreased following 15 h after histidinol treatment. At 24 h, ALT1 mRNA levels dropped to 50% of the values observed in non-treated cells (Fig. 4A). We further tested whether expression of ALT1 and ALT2 is influenced by endoplasmic reticulum (ER) stress. For that purpose, HepG2 cells were incubated with the N-glycosylation inhibitor tunicamycin (TM; 5 μM TM dissolved in 0.1% DMSO). qRT-PCR analysis showed that TM similarly increased ATF4 and ALT2 mRNA levels, reaching a maximum (3-fold induction) 6 h after treatment compared to control cells (incubated with 0.1% DMSO in the absence of TM). Thereafter, mRNA levels for both genes gradually decreased (Fig. 5A). Consistently, ATF4 and ALT2 protein levels increased about 2-fold and 1.5-fold, respectively, when measured 6 h after TM treatment (Fig. 5B). In contrast, ALT1 mRNA levels did not change significantly during the first 8 h of treatment, and declined to 50% of the value observed in control cells at 15 h and 24 h (Fig. 5A).

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3.6. Up-regulation of ALT2 in response to histidinol and tunicamycin in HepG2 cells

Fig. 3. ATF3 and CHOP prevent the induction of ALT2 promoter promoted by ATF4. HepG2 cells were transfected with the promoter constructs pGALT2 − 1534, pGALT2 − 497, pGALT2 − 172, pGALT2 − 90, pGALT2 + 195, pGALT2 − 1534mut or pGL3-Basic, with or without an expression plasmid encoding ATF4, ATF3 and CHOP transcription factors. The promoter activity displayed by the constructs alone was set at 1. Results presented are means ± SD from two independent duplicate experiments.

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Gene specific mRNA/ 18s mRNA

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Fig. 4. Effect of histidinol on ALT2, ALT1 and ATF4 in HepG2 cells. (A). Average value of two independent real-time PCR assays performed on total RNA isolated from HepG2 cells at time 0, 3 h, 6 h, 8 h, 16 h and 24 h after histidinol (2 mM) treatment is shown. ALT2, ALT1 and ATF4 mRNA levels relative to ribosomal 18 s mRNA are expressed as mean ± SD from two independent duplicate or triplicate experiments. Statistical significance related to the corresponding control is indicated as follows: *P b 0.05; **P b 0.01; ***P b 0.001. (B). ATF4 and ALT2 protein levels detected by Western blot using nuclear or total protein extracts, respectively, of control HepG2 cells or 6 h after 2 mM histidinol treatment.

3.7. ATF4-siRNA down-regulates ALT2 and prevents the effect of stress factors on ALT2 expression in HepG2 and Huh-7 cells To get further knowledge on the molecular implication of ATF4 in mediating transcriptional up-regulation of ALT2 in cells under amino acid deprivation or ER stress conditions, the ATF4 expression was knocked-down using siRNAs. In HepG2 cells, transfection with an siRNA targeting ATF4 (si ATF4) significantly decreased the mRNA levels

Gene specific mRNA/ 18s RNA

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Fig. 5. Effect of TM on ALT2, ALT1 and ATF4 in HepG2 cells. (A). Average value of two independent real-time PCR assays performed on total RNA isolated from HepG2 cells at time 0, 3 h, 6 h, 8 h, 16 h and 24 h after TM (culture medium with 5 μM TM in 0.1% DMSO) treatment is shown. ALT2, ALT1 and ATF4 mRNA levels relative to ribosomal 18 s mRNA are expressed as mean ± SD from two independent duplicate or triplicate experiments. Statistical significance related to the corresponding control is indicated as follows: *P b 0.05; **P b 0.01; ***P b 0.001. (B). ATF4 and ALT2 protein levels detected by Western blot using nuclear or total protein extracts of HepG2 cells 6 h after TM treatment (culture medium with 5 μM TM in 0.1% DMSO) and non-treated HepG2 cells (culture medium with 0.1% DMSO).

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of ATF4 and ALT2 to about 35% and 63%, respectively, of the values observed in control cells (transfected with an siRNA with a nonsense/ scrambled sequence; si scr) (Fig. 6A). Treatment of HepG2 cells with either 2 mM histidinol (5 h) or 5 μM TM in 0.1% DMSO (6 h), resulted in a greater knock-down effect on both ATF4 and ALT2 genes. The effect of transfecting siRNA targeting ATF4 over ALT1 expression was also studied. Knock-down of ATF4 promoted a significant increase in ALT1 expression regardless of histidinol or TM treatment (Fig. 6A). Similar results were obtained using another human hepatic cell line, Huh-7. As in HepG2 cells, 5 h of incubation in the presence of 2 mM histidinol and 6 h of treatment with 5 μM TM in 0.1% DMSO induced ATF4 and ALT2 expression, about 1.5- to 2-fold, somewhat less than in non-transfected cells (Fig. 5) probably due to a low cytotoxic effect of the transfection reagent and a concomitant moderate rise of ATF4 levels. ATF4 silencing significantly decreased ATF4 (to about 25% of the values in control cells) and ALT2 (to about 80%) mRNA levels. Treatment of Huh-7 cells with either histidinol or TM, resulted in a greater knockdown effect on both ATF4 and ALT2 genes. In contrast to HepG2 cells, ALT1 mRNA levels in Huh-7 cells remained unchanged irrespective of treatment (Fig. 6B). 4. Discussion Multicellular organisms respond to stress, and nutritional and metabolic changes associated with diseases by triggering different adaptive mechanisms. Among them, the transcriptional control of genes encoding enzymes involved in amino acid metabolism represents an important issue. An increasing number of studies are devoted to elucidate whether ALT isoforms, ALT1 and ALT2, contribute differently to the elevation of ALT activity associated with several pathologies. Recently, Rafter et al. [47] showed that under three different conditions of liver damage (non-alcoholic fatty liver disease, hepatitis C and liver surgery) the leakage of ALT1 activity into plasma greatly exceeded that of ALT2 in humans. Contrary, during skeletal muscle injury, the leakage of ALT2 exceeded that of ALT1 and the proportion of circulating ALT isoforms changed accordingly. Changes in ALT isoforms in plasma reflected relative contents of ALT1 and ALT2 activities in the human liver and skeletal muscle [5]. Regarding ALT regulation, in fatty livers of obese mice, Jadhao et al. [4] found that ALT2 expression increased 2-fold, while ALT1 remained unaffected. The authors suggested that ALT2 could be responsible for the 30% increased ALT activity observed in mice with steatosis. However, in another study, both ALT1 and ALT2 increased in the liver of mice induced liver steatosis by a deficient methionine–choline diet [48]. In a study performed by Reagan et al. [49] the authors found that administration of dexamethasone elevated total ALT activity in liver of mice as a result of increased ALT2 expression. Yang et al. [3] studied the effect of carbon tetrachloride and acetaminophen, two molecules known for their hepatotoxic effect on murine serum ALT. Contribution of ALT1 and ALT2 proteins to the rise in ALT activity in serum depended on the compound analysed: administration of carbon tetrachloride increased ALT1 and ALT2 proteins 4.8- and 3.9-fold, respectively, whereas acetaminophen elevated ALT1 and ALT2 3- and 15-fold, respectively. Despite these efforts, it is necessary to gain more insight into the specific regulation of ALT isoforms to unravel the role of ALT genes in the cell response to stress factors. To study the transcriptional regulation of ALT2, we isolated and characterized the promoter region of ALT2 from human genomic DNA. Transient transfection of human hepatoma HepG2 cells with fusion constructs containing sequential 5′-deletions of the isolated fragment allowed us to conclude that the promoter region within 1534 bp upstream from the transcription start site harbours a functional promoter. Due to the presence of a putative ATF4 binding site and the fact that there is increasing evidence for the involvement of ATF4 in the transcriptional control of several regulatory enzymes of amino acid metabolism [26,37,39,44,45], we addressed whether ATF4 could play a role in the transcriptional control of ALT2. In this study, we demonstrated

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A Two-way ANOVA

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0.0 si scr

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'

Fig. 6. Effect of ATF4 knock-down on the expression of ALT2 and ALT1 genes in HepG2 and Huh-7 cells. (A). HepG2 cells were transfected with either negative control siRNA (si scr) or ATF4 siRNA (si ATF4). After transfection, cells were incubated in the presence or absence of 2 mM histidinol for 5 h or TM (culture medium with 5 μM TM in 0.1% DMSO) for 6 h. To study the effect of TM, control cells in the absence of TM were incubated in 0.1% DMSO. Total RNA was isolated and subjected to qRT-PCR analysis of ALT2, ALT1 or ATF4 mRNA. The values represent mean ± SD of two independent experiments performed in duplicate. (B). Huh-7 cells were transfected with either control siRNA (si scr) or ATF4 siRNA (si ATF4). After transfection, cells were incubated in the presence or absence of 2 mM histidinol for 5 h or TM (culture medium with 5 μM TM in 0.1% DMSO) for 6 h. Total RNA was isolated and subjected to qRT-PCR analysis of ALT2, ALT1 or ATF4 mRNA. The values represent the means ± SD of two independent experiments performed in duplicate. Statistical significance for independent variables (si: transfection with “si ATF4” vs. transfection with “si scr”; and Treatment: incubation with “HISTIDINOL” or “TUNICAMYCIN” vs. control non-treated cells, “-” and “DMSO”, respectively) and of the interaction between independent variables (Interaction) is indicated as follows: *P b 0.05; **P b 0.01; ***P b 0.001; NS, not significant.

that ATF4 confers an activating signal by binding to the CARE element located between −425 and −417 bp upstream from the transcription start site of the human ALT2 gene promoter. We found that mutations in the CARE element abolished the transcriptional activation induced by ATF4. In contrast, ATF4 did not transactivate the ALT1 gene promoter, suggesting that ATF4 plays a specific role on ALT2 promoter regulation. Interestingly, the ATF4 binding sequence motive present in the human ALT2 promoter is found in the putative promoter region of murine ALT2 (data not shown). The time course of ATF4-mediated activation of genes containing CARE elements indicated that ATF4 binds to and transactivates the expression of asparagine synthetase and arginine/lysine transporter cat1 during the first 6 h following stress induction. However, after 6 h, ATF4 binding to DNA gradually decreases, as well as the binding of transcription factors up-regulated by ATF4, such as C/EBPβ, ATF3 and CHOP [50–52]. Our results showed that ATF3 and CHOP completely prevented ATF4-dependent transcriptional activation of the ALT2 gene reporter constructs containing the CARE element (pGALT2 − 1534 and pGALT2 − 497). In mammals, the response to different stress signals, such as lack of oxygen or amino acid deprivation includes phosphorylation of eIF2 by different kinases (PERK, PKR, GCN2 and HRI). Together with the 40S

ribosomal subunit and other translation initiation factors, eIF2 participates in the assembly of the 43S preinitiation complex and bound to GTP and Met-tRNAmet facilitates recognition of the initiation codon, a process that leads to hydrolysis of GTP and the release of GDP-bound eIF2 from the preinitiation complex. Exchange of GDP for GTP by eIF2B allows returning of eIF2 to its activated state. Phosphorylation of the α subunit of eIF2 inhibits eIF2B activity and thus recycling of GDP to GTP on eIF2, leading to a general repression of transcription and increased expression of genes involved in the integrated stress response such as ATF4 [53]. Decreased levels of functional eIF2 due to eIF2B inhibition by phospho-eIF2α results in ribosome scanning bypassing of uORF2, an upstream open reading frame functional under non-stressed conditions that is located at 5′ and overlaps, out of frame, to the ATF4 coding sequence. As a result, cellular stress conditions selectively enhance synthesis of ATF4 protein [31]. In turn, ATF4 promotes the transcription of genes related to metabolism and nutrient uptake, the redox status of the cell, and regulation of apoptosis in order to prevent stress damage. Bearing in mind that ATF4 transactivates the human ALT2 promoter and that it is involved in the transcriptional control of regulatory enzymes in amino acid metabolism and in the cell response to stress, we addressed the effect of amino acid deprivation and ER stress on the expression of ALT isozymes in two hepatic cell lines, HepG2 and Huh-7.

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To analyse the effect of amino acid deprivation, the cells were cultured in the presence of histidinol. Histidinol activates GCN2 kinase by uncharged transfer RNA (tRNAs) and thus leads to eIF2α phosphorylation and increased ATF4 expression [53]. Our results revealed that histidinol treatment up-regulated ALT2 and ATF4 expression in HepG2 and Huh-7 cells. Consistently, ALT2 up-regulation in response to histidinol was significantly impaired by ATF4 knock-down in both HepG2 and Huh-7 cells. In striking contrast to ALT2, ALT1 mRNA levels decreased significantly after 15 h of histidinol treatment in HepG2 cells. Since ATF4 does not transactivate ALT1 gene promoter, downregulation of ALT1 expression by amino acid deprivation would result from the general transcription repression due to eIF2α phosphorylation and eIF2B inhibition under stressful conditions. In the present study we then examined the effect of the ER stressor TM on the expression of ATF4, ALT2 and ALT1. ER stress causes a signal transduction cascade leading to activation of PERK, phosphorylation of eIF2α and increase in ATF4 levels [31,53]. Our experiments performed on HepG2 cells and Huh-7, showed that treatment with TM induced the ATF4 and ALT2 proteins and mRNA levels. Similarly as it was observed with histidinol, ALT1 expression remained unaffected during the first 8 h following TM treatment. Thereafter, ALT1 exhibited decreased mRNA levels at 15 h and 24 h after addition of TM to HepG2 cells. ATF4 silencing significantly impaired ALT2 upregulation in response to TM in both HepG2 and Huh-7 cells. Consistent with our findings, Gentile et al. [54] associated liver injury resulting from TM treatment to increased plasma ALT activity and hepatic lipid content. Altogether, our findings suggest a specific role of ALT2 in the response of cells to stress factors. Increases in ALT2 activity can lead to a rise in the levels of glutamate and pyruvate. In cell stressful conditions glutamate could be used for glutathione synthesis, a key metabolite that prevents damage during oxidative stress, and pyruvate would be derived to obtain energy through the Krebs cycle or used for the synthesis of glucose. In this regard, based on kinetic analysis of ALT isoenzymes Glinghammar et al. [7] concluded that at lower concentrations of substrates, ALT2 is more active than ALT1. The authors suggested that ALT2 may function as a complementary system to generate pyruvate for gluconeogenesis during starvation. Indeed, Mendes-Mourao et al. [55] and Quistorff and Grunnet [56] showed that alanine can be transaminated to provide pyruvate for gluconeogenesis in the mitochondria of rat hepatocytes at levels high enough to maintain blood glucose levels. Furthermore, Reagan et al. [49] indicated the induction of ALT2 by dexamethasone whereas ALT1 was not affected, supporting the notion that ALT2 may be responsible of pyruvate formation under gluconeogenic conditions. In our study we have shown that CHOP suppresses ATF4-mediated up-regulation of ALT2 transcription. A similar mechanism was described by Bouman et al. [57], who reported that CHOP and c-Jun act as suppressors of parkin expression, a protein up-regulated by ATF4 following ER stress via PERK. CHOP is a pro-apoptotic transcription factor induced by eIF2α phosphorylation that links insurmountable levels of ER stress to the cell death machinery [58,59]. Conceivably, in an initial phase of ER stress, ATF4-dependent activation of ALT2 promoter will lead to increased ALT2 activity. However, severe and prolonged ER stress would induce CHOP expression to a threshold level enough to displace ATF4 from the ALT2 CARE element through a preferential binding of CHOP to the ALT2 promoter and to suppress cytoprotective pathways and favour the elimination of irreversibly damaged cells by proapoptotic pathways. In this light, our findings support the notion that ALT2 expression may have a major contribution to the initial phase of metabolic adaptation of the cell to stress. In summary, we have identified ATF4 as a transcriptional activator of human ALT2 gene. Further, we demonstrated ATF4dependent up-regulation of ALT2 by amino acid deprivation and ER stress, suggesting an important role of ALT2 in the initial cell response to metabolic stress.

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Activating transcription factor 4 mediates up-regulation of alanine aminotransferase 2 gene expression under metabolic stress.

Alanine aminotransferase (ALT) provides a molecular link between carbohydrate and amino acid metabolism. In humans, two ALT isoforms have been charact...
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