590 (2016) 177–185

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Gene

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Research paper Intermedin/adrenomedullin 2 is a stress-inducible gene controlled by activating transcription factor 4

Irina E. Kovaleva a, Alisa A. Garaeva b,c, Peter M. Chumakov c,⁎, Alexandra G. Evstafieva a,b,⁎⁎ a Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119992 Moscow, Russia b Department of Bioengineering and Bioinformatics, Lomonosov Moscow State University, 119992 Moscow, Russia c Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilova Street 32, Moscow 119991, Russia article info abstract

Article history: Intermedin or adrenomedullin 2 is a set of -related peptides with a putative tumor angiogenesis pro- Received 26 March 2016 moting activity that are formed by proteolytic processing of the ADM2 gene product. It has been proposed that Received in revised form 3 June 2016 the ADM2 gene is regulated by the estrogen response element (ERE) and hypoxia response elements (HRE) Accepted 17 June 2016 found within its promoter region. In the present study we reveal a functional mechanism by which ADM2 partic- Available online 18 June 2016 ipates in the unfolded response (UPR) and in responses to the mitochondrial respiration chain inhibition. We show that the ADM2 gene is controlled by activating transcription factor 4 (ATF4), the principal regulator of Keywords: Intermedin/adrenomedullin 2 the integrated stress response (ISR). The upregulation of ADM2 mRNA could be prevented by the pharmacolog- Tumor suppressor p53 ical ISR inhibitor ISRIB and by the downregulation of ATF4 with specific shRNA, while ectopic expression of ATF4 Transcription factor ATF4 cDNA resulted in a notable increase in ADM2 gene transcription. A potential ATF4-binding site was identified in Mitochondrial respiratory chain complex the coding region of the ADM2 gene and the requirement of this site during the ATF4-mediated ADM2 gene Integrated stress response promoter activation was validated by the luciferase reporter assay. Mutagenesis of the putative ATF4-response Angiogenesis element prevented the induction of luciferase activity in response to ATF4 overproduction, as well as in response Unfolded protein response to mitochondrial electron transfer chain inhibition by piericidin A and ER stress induction by tunicamycin and brefeldin A. Since ADM2 was shown to inhibit ATF4 expression during myocardial ER stress, a feedback mechanism could be proposed for the ADM2 regulation under ER stress conditions. © 2016 Elsevier B.V. All rights reserved.

1. Introduction (RAMP1–3) (Roh et al., 2004). Human ADM2 gene is located on 22 and encodes a prepropeptide of 148 amino acid Intermedin or adrenomedullin 2 encoded by the ADM2 gene is a set residues (Ni et al., 2014), which is then proteolytically cleaved to yield of multifunctional biologically active peptides belonging to calcitonin bioactive peptides of 53, 47 and 40 amino acid residues. ADM2 is highly family hormones (Roh et al., 2004; Takei et al., 2004). The family in- conserved between different animal species. Accumulating evidences cludes calcitonin, calcitonin gene-related peptide (CGRP), amylin, and indicate that ADM2 is a vital bioactive peptide maintaining vascular ho- adrenomedullin (ADM) and play important roles in maintaining the meostasis. It participates in the regulation of blood pressure and cardiac body homeostasis (Roh et al., 2004). ADM2 binds to receptor complexes function, accelerating angiogenesis, protecting endothelial barrier common to CGRP and ADM, which consist of calcitonin receptor-like function, fighting oxidative and endoplasmic reticulum (ER) stresses receptor (CLR) and one of the three receptor activity-modifying (Ni et al., 2014). ADM2 is involved in the pathogenesis of such common vascular diseases as hypertension, vascular calcification and atheroscle- rosis (Ni et al., 2014). Abbreviations: ADM, adrenomedullin; Akt, serine/threonine kinase, protein kinase B; The induction of ADM2 expression was shown to take place in many ATF4, activating transcription factor 4; Erk1/2, extracellular signal–regulated kinases 1/2; cases of cardiovascular pathophysiology. In particular, high ADM2 α α eIF2 , subunit of eukaryotic initiation factor 2; ER stress, endoplasmic reticulum mRNA expression levels were observed in leukocytes of patients with stress; FDR, false discovery rate; ISR, the integrated stress response; ISRIB, inhibitor of the ISR; MAPK, mitogen-activated protein kinase; PI3K, phosphoinositide 3-kinase; PKA, chronic heart failure (Cabiati et al., 2014). Plasma levels of ADM2 were protein kinase A; shRNA, short hairpin RNA; UPR, unfolded protein response; VEGF, shown to be increased in patients with severe lesions in coronary arter- vascular endothelial growth factor. ies and acute myocardial infarction (Lv et al., 2013). The upregulation of ⁎ Corresponding author. ADM2 in cardiovascular pathologies is likely to play a protective role (Ni ⁎⁎ Correspondence to: A.G. Evstafieva, Belozersky Institute of Physico-Chemical Biology, et al., 2014). It has been suggested that through the PI3K/Akt or cAMP/ Lomonosov Moscow State University, 119992 Moscow, Russia. E-mail addresses: [email protected] (P.M. Chumakov), PKA and MAPK/Erk1/2 pathways ADM2 attenuates the ER stress and in- [email protected] (A.G. Evstafieva). duces protective autophagy in the myocardium (Chen et al., 2013; Teng

http://dx.doi.org/10.1016/j.gene.2016.06.037 0378-1119/© 2016 Elsevier B.V. All rights reserved. 178 I.E. Kovaleva et al. / Gene 590 (2016) 177–185 et al., 2011). However, in cancer the increased ADM2 expression might fragment of the coding region was amplified by the PCR on a total promote the disease. High ADM2 mRNA expression levels were ob- DNA from HCT116 cells with primers Adm2_forward (5′-AGGTACCCA served in hepatocellular (Guo et al., 2012), and colorectal carcinomas GGCTCGGGGCTGCGTCC-3′) and Adm2_reverse (5′-GAGAAGCTTG (Hikosaka et al., 2011) suggesting a role for ADM2 in cancer angiogene- AGGAGGCTGATGCAACCCAG-3′). The gel-purified fragment was ligated sis where in cooperation with VEGF it helps building a functional vascu- into KpnI-HindIII sites of the pUC19 plasmid for sequencing. The lature supporting tumor growth (Zhang et al., 2012). sequence-verified insert was transferred as a KpnI-HindIII/Klenow frag- The expression of the ADM2 gene was shown to be induced by many ment into the plasmid pGL3-Basic (Promega, WI) processed with NcoI/ different influences, including bacterial lipopolysaccharide (LPS) in a rat Klenow and KpnI. testis (Li et al., 2013), thyroid stimulating hormone in the rat thyroid gland (Nagasaki et al., 2014), docetaxel treatment of non-small cell 2.1.2. pGL3-ADM2 (−383, +169)_mut lung cancer cell line (Che et al., 2013), estrogen exposure of pituitary The reporter with the mutated putative ATF4 binding site was ob- cells (Lin Chang et al., 2005) and hypoxic conditions in the lungs and tained in the same way except the mutagenizing primer Adm2_pr- cell lines of mouse origin (Pfeil et al., 2009). Studies on molecular mech- mut (5′-GAGAAGCTTGAGGAGGCTTATTCTACCCAGGG-3′) was used in- anisms of ADM2 expression induction after exposure to estrogens and stead of Adm2_reverse primer. hypoxia have revealed a consensus estrogen response element (ERE) and several putative hypoxia response elements (HRE) within 2.7 kb 2.1.3. pGL3-ADM2 (−383, +124) upstream region of the gene, both in human and mouse. By luciferase To obtain a reporter with ATF4-binding site deleted, the 507 bp DNA reporter assay it was shown that estrogen treatment of mouse pituitary fragment comprising the ADM2 promoter region and 5′-untranslated cells resulted in a stimulation of the Adm2 gene promoter (Lin Chang region with intron was PCR-amplified on HCT116 DNA with primers et al., 2005). Also, an ectopic expression of HIF-1α in HEK293T cells re- Adm2_forward and Adm2_rev_delta (5′-GAGAAGCTTGGCGGGCGGGG sulted in a dose-dependent increase in ADM2 gene promoter activity, CTGCAGG-3′). The gel-purified fragment was ligated into KpnI-HindIII suggesting that the stimulation of ADM2 expression by hypoxia was me- sites of pUC19 plasmid for sequencing and then transferred into the diated by HIF-1α (Pfeil et al., 2009). same sites of pGL3-Basic. In the present study we reveal an additional mechanism by which ATF4-expressing plasmid pHM-ATF4 has been previously described ADM2 responds to stresses. We show that the ADM2 gene is controlled (Garaeva et al., 2016). by activating transcription factor 4 (ATF4), the principal regulator of the integrated stress response (ISR). ATF4 integrates signals from four 2.2. Cell lines and chemicals distinct protein kinases that are activated in response to diverse cellular stresses. These are PERK (PKR-like endoplasmic reticulum kinase) acti- The human carcinoma cell lines HCT116 and HeLa were grown in vated in response to overloading of ER with misfolded or excessively DMEM, containing 10% fetal calf serum (HyClone, UT) at 37 °C, 5% CO2 synthesized proteins, GCN2 (general control non-depressible 2 kinase) to 50–70% confluence. For respiratory chain inhibition we treated the – in response to nutrient deprivation, PKR (protein kinase R) – in re- cells with 1 μM myxothiazol or 2 μM piericidin A (Sigma-Aldrich, MO) sponse to viral infection and HRI (heme-regulated inhibitor kinase) – for indicated in the figure legends periods of time. Both inhibitors in erythrocytes by low levels of heme (Baird and Wek, 2012; were added in quantities necessary to block respiration of HeLa cells Wengrod and Gardner, 2015). These protein kinases induce the phos- (Shchepina et al., 2002). 1 mM uridine was added to the culture medi- phorylation of eIF2α (eukaryotic translation initiation factor 2, α sub- um to prevent p53 activation when indicated. ISRIB (Sigma-Aldrich, unit) at Ser51 leading to a global attenuation in translation. However, MO) was added to the final concentration 200 nM as recommended in the block results in a preferable translation of a set of mRNAs containing (Sidrauski et al., 2013). For induction of ER stress the cells were treated small upstream open reading frames (uORFs), which includes the ATF4 with 1 μg/ml tunicamycin or 0.5 μg/ml brefeldin A (Sigma-Aldrich, MO) gene mRNA (Baird and Wek, 2012; Wengrod and Gardner, 2015; Lu for 14 h. For ATF4 ectopic expression, the cells in 60 mm dish were et al., 2004). ATF4 activates transcription of certain involved in transfected with 2 μg pHM-ATF4 using the TurboFect transfection re- synthesis and folding of proteins, transport of nutrients, metabolism, agent (ThermoFisher Scientific, MA) as recommended by the manufac- redox regulation and apoptosis. As ATF4 is a common downstream tar- turer. The quantity of plasmid DNA was brought to 6 μgbythe“empty” get of different eIF2-specific kinases, the effects produced by activated vector pcDNA4/HisMax/B (Invitrogen, CA) used for pHM-ATF4 plasmid eIF2α/ATF4 pathway are often referred to as ISR. In response to environ- construction (Garaeva et al., 2016). Lentiviral constructs expressing mental stresses ATF4 can also be induced at transcription level (Dey ATF4 shRNA or scrambled shRNA were prepared using lentiviral vector et al., 2010). It is assumed that the regulation of ATF4 at transcriptional pLSLP (Chumakov et al., 2010) and introduced into cells as described and translational levels results in modulation of the expression of key (Evstafieva et al., 2014). regulatory genes during the ISR (Dey et al., 2010). We have found that in human cancer cell lines ADM2 mRNA expres- 2.3. analysis sion is induced in response to mitochondrial respiration chain inhibition and ER stress. A potential ATF4-binding site was identified in the coding The RNA-seq data (Evstafieva et al., 2014) deposited in NCBI region of ADM2 gene and the requirement of this site for the ATF4- (BioProject accession number SRP043021) were used to identify enrich- mediated ADM2 gene promoter activation was validated by luciferase ment with Gene Ontology term “angiogenesis” by DAVID gene function- reporter assay. Since ADM2 was shown to inhibit ATF4 expression dur- al annotation tool (Huang da et al., 2009). ing myocardial ER stress (Teng et al., 2011), a feedback mechanism could be proposed for the ADM2 regulation under ER stress conditions. 2.4. Reporter assays

2. Materials and methods HeLa cells in 12-well plates were transfected essentially as de- scribed previously (Garaeva et al., 2016). The following plasmids 2.1. Plasmid construction were introduced into the each well of the culture plate: one of the re- porter plasmids, pGL3-ADM2 (−383, +169), pGL3-ADM2 (−383, 2.1.1. pGL3-ADM2 (−383, +169) +124), or pGL3-ADM2 (−383, +169)_mut (0.1 μg), the vector The reporter containing the putative ATF4 binding site was obtained pHM-ATF4 (0.5 μg) for ATF4 ectopic expression, and the normaliza- in the following way. The 552 bp DNA fragment comprising the ADM2 tion plasmid pcDNA4/HisMax/lacZ (Invitrogen, CA) (0.5 μg). The promoter region, 5′-untranslated region with intron and 45 bp long quantity of plasmid DNA was brought to 2 μgbythe“empty” vector I.E. Kovaleva et al. / Gene 590 (2016) 177–185 179 pcDNA4/HisMax/B used for pHM-ATF4 plasmid construction 3. Results (Garaeva et al., 2016). 44 h after transfection the cells were proc- essed for measurements of the luciferase and β−gal activities as de- 3.1. Coordinated ADM2 and ATF4 expression in response to mitochondrial scribed (Garaeva et al., 2016). The means and standard deviations dysfunction were calculated on the basis of at least three independent experi- ments. Statistical significancewasdeterminedbytheStudent'st- In the previous study (Evstafieva et al., 2014) we tested the response of test (*P b 0.05, **P b 0.01, ***P b 0.001). human cells to stresses induced by an inhibition of the mitochondrial respi- ration chain. The transcriptome changes following a treatment of HCT116 2.5. Real-time PCR colon carcinoma cell line with the complex III inhibitor myxothiazol were monitored by mRNA-seq. It was discovered that a short-term (5 h) inhibi- Isolation of the total RNA, cDNA synthesis and real time PCR were tion of complex III resulted in induction of expression of ATF4, the principal carried out as described (Evstafieva et al., 2014). The following primers regulator of the ISR, and its transcriptional targets, while the longer (13 h) were used: ADM2_dir 5′-CGTAGAAGGTAGAATAAGTGG-3′, ADM2_rev inhibition led to p53 tumor suppressor activation, which inhibited the ATF4 5′-TGAGAAGCAACTGTGAGG-3′;ATF4_dir5′-CTTCACCTTCTTACAACC gene transcription and reversed the upregulation to downregulation TCTTC-3′, ATF4_rev 5′-GTAGTCTGGCTTCCTATCTCC-3′; 18S_dir 5′- (Evstafieva et al., 2014). The long term inhibition of respiration chain com- CGGACAGGATTGACAGATTG-3′,18S_rev5′-CAGAGTCTCGTTCGTTAT plex III induced the activation of p53 as the result of an impairment of the CG-3′; CHOP_dir 5′-CCTGCTTCTCTGGCTTGG-3′, CHOP_rev 5′-CTTGGT de novo pyrimidine biosynthesis pathway (Khutornenko et al., 2010). Ab- CTTCCTCCTCTTCC-3′. The reference 18S rRNA was used for normaliza- rogation of the p53 activation by uridine supplementation prevented the tion. The means and standard deviations were calculated on the basis dropdown of the elevated mRNAs for ATF4-responsive genes during the of at least three independent experiments. Statistical significance long-term (13 h) treatment with myxothiazol (Evstafieva et al., 2014). was determined by the Student's t-test (*P b 0.05, **P b 0.01, The list of 165 genes with a similar regulation mode was identified (Sup- ***P b 0.001). plementary Tables 1 and 2 in Evstafieva et al., 2014). The list contained N30 known ATF4-target genes and provided a set of genes for a search of 2.6. Western analysis potential new ATF4 transcription targets. We selected from this list a dozen of candidate genes most significantly upregulated by myxothiazol For immunoblotting the cells were lysed in reporter lysis buffer in the absence of p53 activation and downregulated after p53 activation (Promega, Inc.). 50 μg aliquots of total protein were fractionated by (ANGPT4, ADM2, HKDC1, KRT16, FAM129A, MDGA1, VGF, PDCD4, КRT6A, 12% SDS-polyacrylamide gel electrophoresis and processed as described JDP2), and then investigated the effect of ATF4 overproduction in HeLa earlier (Khutornenko et al., 2014). Antibodies to p53 (DO-1) and and HCT116 cells on the expression of these genes by qRT-PCR. beta-actin (C-2) diluted at a ratio of 1:500 were from Santa Cruz Among the candidate genes ADM2 was induced to the greatest ex- Biotechnology, CA. tent in response to the overexpression of ATF4 (Fig. 1). Overproduction

Fig. 1. mRNA levels of potential ATF4 target genes in response to ATF4 overexpression. ATF4 mRNA levels in HeLa (a) and HCT116 (c) cells transfected with ATF4-expressing plasmid pHM- ATF4 (ATF4) or with the empty vector pcDNA4/HisMax/B (control). mRNA fold changes of the indicated genes in HeLa (b) and HCT116 (d) cells transfected with ATF4-expressing plasmid pHM-ATF4. The data was obtained by qRT-PCR as described in M&M. 180 I.E. Kovaleva et al. / Gene 590 (2016) 177–185 of ATF4 resulted in an 11-fold increase in ADM2 mRNA levels in HeLa We hypothesized that expression of ADM2 in response to mito- cells (Fig. 1a, b) and a 3.5-fold increase in HCT116 cells (Fig. 1c, d), chondrial dysfunction was regulated by ATF4, a principal regulator identifying ATF4 as the major upstream regulator of ADM2.Taking of the ISR. To check this hypothesis, we studied the effect of the inte- into account the important biological activities of adrenomedullin 2 as grated stress response inhibitor ISRIB and low ATF4 levels on ADM2 well as insufficiently studied regulation of its gene, ADM2 was chosen gene transcription. for further investigation. The mRNA-seq results concerning a coordinated regulation of 3.2. Effect of the integrated stress response inhibitor ISRIB and low ATF4 ADM2 and ATF4 genes as a result of the electron transfer chain inhi- levels on ADM2 gene expression bition were confirmed by qRT-PCR analysis. At the early time point (5 h) of the myxothiazol treatment the levels of ADM2 and ATF4 ISRIB, a recently identified pharmacological inhibitor of ISR, was mRNAs were increased (Fig. 2a, d) and then by 13 h dropped shown to act downstream of all eIF2-kinases (Sidrauski et al., 2013). below the control level (Fig. 2b, e). Supplementation of cells with Phosphorylation of eIF2α reduces protein biosynthesis by inactivation uridine abrogated the myxothiazol-induced upregulation of p53 of eIF2B, a guanine nucleotide exchange factor that is responsible for (Fig. 2h) and the elevated expression of the p53 target gene the exchange of GDP for GTP in the eIF2 complex. After each round of CDKN1A (Fig. 2g). Besides, it largely reversed the ATF4 mRNA translation initiation, the eIF2·GDP complex is released, and eIF2 must downregulation at 13 h of myxothiazol exposure (Fig. 2e), as ex- then be reloaded with GTP to enter a next round of ternary complex for- pected. The upregulation of ADM2 mRNA was also observed in re- mation. When phosphorylated at Ser-51, eIF2α dissociates more slowly sponse to 13 h treatment with myxothiazol and uridine (Fig. 2b). from the eIF2B and locks eIF2B in an inactive state. As eIF2 is more The similar ADM2 and ATF4 mRNA induction was found in abundant than eIF2B, a small fraction of phosphorylated eIF2α is suffi- response to complex I inhibitor piericidin A (Fig. 1c, f), which did cient to sequester a large part of available eIF2B, leading to a substantial not induce p53. reduction in overall protein synthesis. ISRIB was shown to stabilize ADM2 and ATF4 mRNAs were also coordinately induced in response eIF2B, increase its guanine nucleotide exchange factor activity and to inhibition of mitochondrial electron transfer chain by myxothiazol counteract the negative effect of eIF2α phosphorylation on translation and piericidin A in cervical carcinoma cell line HeLa (Fig. 3), indicating (Sekine et al., 2015; Sidrauski et al., 2015). As a result, ISRIB restores that it was not a cell line specificeffect. the overall cellular translation, but specifically reduces translation of

Fig. 2. Coordinated regulation of ADM2 and ATF4 mRNAs after the mitochondrial electron transfer chain inhibition in HCT116 cells. ADM2 mRNA levels (a–c), ATF4 mRNA levels (d–f) and p53-responsive CDKN1A mRNA levels (g) after treatment with myxothiazol for 5 h (a, d), with myxothiazol and/or uridine for 13 h (b, e, g), and with piericidin A for 13 h (c, f). Thedatawas obtained by qRT-PCR as described in M&M. (h) Immunoblot analysis of p53 protein levels in HCT116 cells treated for 13 h with myxothiazol or/and uridine as indicated. I.E. Kovaleva et al. / Gene 590 (2016) 177–185 181 certain mRNAs containing upstream open reading frames, including the ATF4 mRNA (Sidrauski et al., 2013). We found that ISRIB suppresses the induction ADM2 transcripts in response to inhibitors of the mitochondrial electron transfer chain myxothiazol and piericidin A (Fig. 4). The result demonstrates a key role of ISR in the upregulation of ADM2 expression during respiration chain inhibition and suggests that ATF4 participates in this process. To confirm this conclusion we decreased the expression of ATF4 by RNA interference in HCT116 and HeLa cells. The cells were infected with lentiviral construct expressing short hairpin RNA (shRNA) directed against the ATF4 mRNA, with the subsequent puromycin selection. As was shown by qRT-PCR, the levels of ATF4 mRNA were significantly de- creased in these cells in comparison to the cells expressing the scram- Fig. 4. ISR is essential for upregulation of ADM2 transcripts in response to the inhibition of bled control shRNA (Fig. 5a, b). It resulted in a statistically significant mitochondrial electron transfer chain. ADM2 mRNA levels in HeLa cells after treatment with 1 μM myxothiazol for 4 h (a) or 2 μM piericidin A for 8 h (b) in the presence or suppression of both the basal and induced levels of ADM2 mRNAs without 200 nM ISRIB, as indicated. The data was obtained by qRT-PCR as described in (Fig. 5c, d). We conclude that the ATF4 transcription factor regulates M&M. transcription of ADM2.

To test whether the identified site may be responsible for the ATF4- 3.3. ATF4-response element in ADM2 gene mediated regulation of ADM2 gene expression we designed a reporter construct (Fig. 6a). We amplified by PCR from genomic DNA a fragment ATF4 is known to regulate transcription by formation of the dimer encompassing the ADM2 promoter region (−383 bp to 0) with down- complexes with the transcription factors of AP-1, Jun and C/EBP stream 5′-UTR with first intron (0 to +124 bp) and a portion of the cod- (CCAAT-enhancer binding protein) families (Horiguchi et al., 2012; ing region (+125 to +169 bp) containing the putative ATF4-binding Kilberg et al., 2009). The dimers bind to hybrid C/EBP-ATF responsive site. The fragment was fused in frame with the firefly luciferase reporter elements (CARE), consisting of two half-sites required for binding of gene in the reporter construct pGL3-ADM2 (−383, +169) that was C/EBP and ATF family proteins, respectively (Kilberg et al., 2009). By further transfected into HeLa cells along with a normalization control scanning ADM2 promoter region, we did not find potential ATF4 binding reporter construct constitutively expressing LacZ gene. We found that sites at reasonable distances upstream of the transcription start site. the ectopic expression of ATF4 resulted in a 3.5-fold increase in lucifer- However a suitable candidate for ATF4-response element (GTTGCA ase activity (Fig. 6b), suggesting that the genome fragment containing TCA corresponding to the consensus XTTXCATCA (Kilberg et al., the putative ATF4 site within the protein-coding region of ADM2 gene 2009)) was found at a distance of 30 bp downstream from the ADM2 was capable of mediating gene activation by ATF4. To validate the translation start codon. hypothesis, the putative ATF4 binding site was either deleted (pGL3-

Fig. 5. ATF4 knockdown leads to ADM2 mRNA downregulation. ATF4 (a, b) and ADM2 Fig. 3. Coordinated regulation of ADM2 and ATF4 mRNA after the mitochondrial electron (c, d) mRNA levels in HCT116 (a, c) and HeLa (b, d) cells expressing either scrambled transfer chain inhibition in HeLa cells. ADM2 mRNA levels (a, b) and ATF4 mRNA levels shRNA from pLSLP-sh-sc construct (sc) or ATF4 shRNA from pLSLP-sh-ATF4 construct (c, d) after treatment with myxothiazol for 4 h (a, c) or with piericidin A for 8 and 14 h (ATF4) after treatment with 2 μM piericidin A for 8 h or 1 μM myxothiazol for 5 h as (b, d). The data was obtained by qRT-PCR as described in M&M. indicated. The data was obtained by qRT-PCR as described in M&M. 182 I.E. Kovaleva et al. / Gene 590 (2016) 177–185

ADM2 (−383, +124) construct) or mutated at three most conserved 3.4. Induction of ADM2 expression in response to ER stress positions by converting the sequence GTTGCATCAintoGTAGAATAA (pGL3-ADM2 (−383, +169)_mut construct). Either of the constructs ATF4 is associated with the PERK/eIF2α/ATF4 pathway of the unfold- (Fig. 6a) was not capable of luciferase induction in response to ectopic ed protein response (UPR)/ER stress (Wek and Cavener, 2007) suggest- expression of ATF4 (Fig. 6b). We conclude that the GTTGCATCA element ing that the ADM2 gene could also respond to ER stresses. We tested located 30 bp downstream of the translation initiation codon within the ADM2 gene expression during UPR and found a substantial increase in ADM2 gene is responsible for the regulation of ADM2-promoter driven ADM2 transcripts following treatments with ER stress inducers expression by ATF4. tunicamycin (blocks N-glycosylation of proteins) and brefeldin A To test whether the stimulation of ADM2 expression following the (inhibits protein transport from ER to the Golgi apparatus) (Fig. 7a). inhibition of mitochondrial respiration chain is mediated by ATF4, The induction of ADM2 expression was associated with an expression we measured changes in the luciferase expression from the reporter of the ER stress markers ATF4 (Fig. 7b) and C/EBP homologous protein plasmids pGL3-ADM2 (−383, +169) and pGL3-ADM2 (−383, (CHOP) (Fig. 7c). +169)_mut transfected into HeLa cells. Treatment of the cells with To test whether the stimulation of ADM2 expression during the ER complex I inhibitor piericidin A resulted in a substantially higher in- stress may be mediated by ATF4, we analyzed the effect of tunicamycin crease in luciferase activity in the cells transfected with the reporter and brefeldin A on the ADM2 gene fragment [−383, +169]-driven re- containing the wild type ATF4 binding site compared to the mutant porter activity in HeLa cells. Both compounds induced expression of the one (Fig. 6c). Together, these results support the conclusion that in reporter gene in cells transfected with the reporter containing the wild response to mitochondrial electron transfer chain inhibition the type ATF4 binding site. Importantly, mutations in the ATF4-binding site ADM2 promoter induction is mediated by the ATF4 transcription completely prevented the increase in luciferase activity induced by factor. brefeldin A and substantially suppressed the increase in luciferase

Fig. 6. Stimulation of the ADM2-promoter driven reporter gene expression in response to ATF4 overproduction and respiration chain inhibition depends on the GTTGCATCA site in ADM2 coding region. (a) Schematic representation of the reporter constructions. (b) The activity of reporters with a wild type (ADM2 −383, +169), deleted (ADM2 −383, +124), or mutated (ADM2 −383, +169_mut) putative ATF4-response element in transfected HeLa cells with overexpression of ATF4. Control cells were transfected with an empty vector. (c) The activity of reporters with a wild type (ADM2 −383 + 169) or mutated (ADM2 −383 + 169_mut) putative ATF4-response element in transfected HeLa cells treated with 2 μM piericidin A for 16 h compared to untreated control cells. The effects were normalized to reporter activities in control cells. I.E. Kovaleva et al. / Gene 590 (2016) 177–185 183

Fig. 7. Upregulation of ADM2 mRNA and the ADM2-promoter driven reporter gene activity in response to ER stress inducers. The levels of ADM2 (a), ATF4 (b), and CHOP (c) mRNAs after treatment HeLa cells with tunicamycin or brefeldin A for 16 h. The data was obtained by qRT-PCR as described in M&M. (d, e) The activity of reporters with a wild type (ADM2 −383, +169) or mutated (ADM2 −383, +169_mut) putative ATF4-response element in transfected HeLa cells treated with tunicamycin (Tm) or brefeldin A (BFA) for 16 h compared to untreated control cells. The effects were normalized to reporter activities in control cells.

activity induced by tunicamycin (Fig. 7d, e). The result indicates that the Table 1 ATF4 transcription factor contributes to ADM2 promoter induction in The list of genes involved in angiogenesis (GO:0001525) upregulated after mitochondrial response to ER stress. respiration chain complex III inhibition with myxothiazol for 5 h in HCT116 cells. Fold changes in mRNA levels according to mRNA-seq data are presented (FDR b 0.05). 4. Discussion Gene DAVID gene name Fold change We have found that ADM2 mRNA is induced in response to inhibi- AIMP1 aminoacyl tRNA synthetase complex-interacting 1.25 tion of the mitochondrial electron transfer chain in human cancer cell multifunctional protein 1 lines. The induction correlated with an upregulation of transcription ANXA2 annexin A2 1.29 ATP5B ATP synthase, H+ transporting, mitochondrial F1 complex, 1.19 from some known ATF4-regulated genes and apparently belonged to beta polypeptide the ISR. The abrogation of ISR by specific small molecule inhibitor CTGF connective tissue growth factor 2.42 ISRIB prevented the ADM2 mRNA induction in response to complex I CYR61 cysteine-rich, angiogenic inducer, 61 2.04 and III inhibitors piericidin A and myxothiazol, respectively. As ISRIB ELK3 ELK3, ETS-domain protein (SRF accessory protein 2) 1.62 selectively suppresses translation of some stress-responsive mRNAs EREG epiregulin 1.61 HMOX1 hemeoxygenase (decycling) 1 1.68 including ATF4 mRNA (Sidrauski et al., 2013), the data suggests a role ID1 inhibitor of DNA binding 1, dominant negative 1.26 for ATF4 transcription factor in the upregulation of ADM2 mRNA in helix-loop-helix protein response to respiration chain inhibitors. IL8 interleukin 8 3.99 We reaffirmed the role of ATF4 in the regulation of ADM2 gene ex- JAG1 jagged 1 (Alagille syndrome) 1.37 JUN Jun oncogene 1.68 pression. Ectopic expression of ATF4 resulted in a substantial increase KLF5 Kruppel-like factor 5 (intestinal) 1.64 in ADM2 mRNA levels. On the contrary, decline of ATF4 expression by MYH9 myosin, heavy chain 9, non-muscle 1.55 RNA interference led to ADM2 mRNA downregulation, suggesting NRP1 neuropilin 1 1.38 ATF4 as a major upstream regulator of ADM2 gene. Our data substanti- PDGFA platelet-derived growth factor alpha polypeptide 2.16 ate a potential mechanism for this effect. Results obtained with tran- PLAU plasminogen activator, urokinase 2.51 PTK2B PTK2B protein tyrosinekinase 2 beta 2.33 scription reporters indicate that ATF4 binding site GTTGCATCA, RHOB ras homolog gene family, member B 1.41 located at a distance of 30 bp downstream of the translation start SCG2 secretogranin II (chromogranin C) 1.34 codon within the ADM2 gene, plays the key role in ATF4-mediated SH2D2A SH2 domain protein 2A 1.91 induction of ADM2 promoter. Mutations in this site prevented the in- SRF serum response factor (c-fos serum response 1.47 element-binding transcription factor) duction of luciferase activity in response to ectopic expression of ATF4, TGFA transforming growth factor, alpha 1.26 as well as in response to electron transfer chain inhibition by piericidin THBS1 thrombospondin 1 2.11 A. TNFRSF12A tumor necrosis factor receptor superfamily, member 12A 1.42 A biological role for the ATF4-mediated upregulation of ADM2 gene VEGFA vascular endothelial growth factor A 1.66 expression in response to mitochondrial dysfunction remains illusive. 184 I.E. Kovaleva et al. / Gene 590 (2016) 177–185

ADM2 is a proangiogenic factor playing a critical role in normal vascular Acknowledgements remodeling as well as in tumor angiogenesis (Zhang et al., 2012). The respiratory chain inhibition appears to be associated with deficiencies We are grateful to Prof. A.B. Vartapetian and Dr. D.E. Andreev for of amino acids (Evstafieva et al., 2014), glucose and energy (ATP). The fruitful discussions. This work was supported by the Russian Foundation shortages supposedly result in activation of the GCN2–eIF2α–ATF4 for Basic Research grant 15-04-04945-a (A.G.E.), and by the Russian pathway of ISR (Evstafieva et al., 2014) that promotes the survival of Science Foundation grant 14-50-00060 (P.M.C.). cancer cells under nutrient deprivation (Ye et al., 2010). The major function of ISR is thought to include activation of a broad translational References and transcriptional program that improves resistance of cells to stress conditions (Baird and Wek, 2012; Wengrod and Gardner, 2015; Lu Afonyushkin, T., Oskolkova, O.V., Philippova, M., Resink, T.J., Erne, P., Binder, B.R., et al., 2010. Oxidized phospholipids regulate expression of ATF4 and VEGF in endothelial et al., 2004; Dey et al., 2010). One possible solution contributing to cells via NRF2-dependent mechanism: novel point of convergence between electro- protection of tissues from the mitochondrial dysfunction stresses philic and unfolded protein stress pathways. Arterioscler. Thromb. Vasc. Biol. 30 – would be an increased blood flow that delivers more nutrients to (5), 1007 1013. http://dx.doi.org/10.1161/atvbaha.110.204354 (Epub 2010/02/27. PubMed PMID: 20185790). stressed cells and takes away more toxic byproducts. Arguing for this Baird, T.D., Wek, R.C., 2012. Eukaryotic initiation factor 2 phosphorylation and translation- scenario is our bioinformatic reanalysis of the transcriptome data al control in metabolism. Adv. Nutr. 3 (3), 307–321. http://dx.doi.org/10.3945/an.112. (Evstafieva et al., 2014) revealing the enrichment of the set of genes 002113 (Epub 2012/05/16. PubMed PMID: 22585904; PubMed Central PMCID: PMCPmc3649462). up-regulated in response to transient (5 h) inhibition of respiration Cabiati, M., Sabatino, L., Svezia, B., Caruso, R., Verde, A., Caselli, C., et al., 2014. chain by myxothiazol with transcripts from the genes involved in Adrenomedullin and intermedin gene transcription is increased in leukocytes of angiogenesis (GO:0001525, FDR 0.0051) (Table 1). Interestingly, a patients with chronic heart failure at different stages of the disease. Peptides 55, 13–16. http://dx.doi.org/10.1016/j.peptides.2014.01.028 (Epub 2014/02/18 PubMed portion of this list (vascular endothelial growth factor A (VEGF-A), PMID: 24531032). cysteine-rich angiogenic inducer 61 (CYR61), epiregulin (EREG), Che, C.L., Zhang, Y.M., Zhang, H.H., Sang, Y.L., Lu, B., Dong, F.S., et al., 2013. DNA microarray Kruppel-like factor 5 (KLF5), interleukin 8 (IL8)) is also included in reveals different pathways responding to paclitaxel and docetaxel in non-small cell lung cancer cell line. Int. J. Clin. Exp. Pathol. 6 (8), 1538–1548 (Epub 2013/08/08. the set of proangiogenic genes recently shown to be up-regulated PubMed PMID: 23923072; PubMed Central PMCID: PMCPmc3726969). by the UPR-inducing drug Thapsigargin (Pereira et al., 2010). There- Chen, H., Wang, X., Tong, M., Wu, D., Wu, S., Chen, J., et al., 2013. Intermedin suppresses fore, our data argue for the involvement of ATF4 in pro-angiogenic pressure overload cardiac hypertrophy through activation of autophagy. PLoS One 8 effects induced by ISR. (5), e64757. http://dx.doi.org/10.1371/journal.pone.0064757 (Epub 2013/06/06. PubMed PMID: 23737997; PubMed Central PMCID: PMCPmc3667197). The ATF4 branch of UPR is recognized as a pathogenic mechanism Chumakov, S.P., Kravchenko, J.E., Prassolov, V.S., Frolova, E.I., Chumakov, P.M., 2010. Effi- for the vascular damage characteristic to diabetes and atherosclerosis cient downregulation of multiple mRNA targets with a single shRNA-expressing – (Afonyushkin et al., 2010). ER stress participates in the pathogenesis lentiviral vector. Plasmid 63 (3), 143 149 (Epub 2010/01/13. doi: S0147- 619X(09)00140-1 [pii] 10.1016/j.plasmid.2009.12.003. PubMed PMID: 20064551; of various cardiovascular diseases such as coronary heart disease, cardi- PubMed Central PMCID: PMC2849729). ac ischemia/reperfusion injury, cardiomyopathy, and heart failure (Teng Dey, S., Baird, T.D., Zhou, D., Palam, L.R., Spandau, D.F., Wek, R.C., 2010. Both transcription- et al., 2011). Therefore, an inhibition of ER stress response pathways al regulation and translational control of ATF4 are central to the integrated stress re- sponse. J. Biol. Chem. 285 (43), 33165–33174. http://dx.doi.org/10.1074/jbc.M110. provides protection for cardiovascular system. We have found that 167213 (Epub 2010/08/25. PubMed PMID: 20732869; PubMed Central PMCID: ATF4 participates in ADM2 mRNA induction under ER stress conditions. PMCPMC2963398). On the other hand, published data indicate that ER stress can be mitigat- Evstafieva, A.G., Garaeva, A.A., Khutornenko, A.A., Klepikova, A.V., Logacheva, M.D., Penin, fi – A.A., et al., 2014. 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