Gene doubling increases glyoxalase 1 expression in RAGE knockout mice

Journal Pre-proof Gene doubling increases glyoxalase 1 expression in RAGE knockout mice

Babett Bartling, Katja Zunkel, Samiya Al-Robaiy, Faramarz Dehghani, Andreas Simm PII:

S0304-4165(19)30224-7

DOI:

https://doi.org/10.1016/j.bbagen.2019.129438

Reference:

BBAGEN 129438

To appear in:

BBA - General Subjects

Received date:

19 July 2019

Revised date:

9 September 2019

Accepted date:

12 September 2019

Please cite this article as: B. Bartling, K. Zunkel, S. Al-Robaiy, et al., Gene doubling increases glyoxalase 1 expression in RAGE knockout mice, BBA - General Subjects(2019), https://doi.org/10.1016/j.bbagen.2019.129438

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© 2019 Published by Elsevier.

Journal Pre-proof Gene doubling increases glyoxalase 1 expression in RAGE knockout mice

Babett Bartling

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, Katja Zunkel 1 , Samiya Al-Robaiy 1 , Faramarz Dehghani 2 , Andreas Simm

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Department of Cardiac Surgery, Middle German Heart Center, University Hospital Halle (Saale), Martin

Luther University Halle-Wittenberg, Halle (Saale), Germany Institute of Anatomy, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany

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Corresponding author: Dr. Babett Bartling, Klinik für Herzchirurgie, Mitteldeutsches Herzzentrum,

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E-mail address: [email protected]

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Universitätsklinikum Halle (Saale), Ernst-Grube-Str. 40, D-06120 Halle (Saale), Germany

Journal Pre-proof Abstract Background The receptor for advanced glycation end-products (RAGE) is a multifunctional protein. Its function as pattern recognition receptor able to interact with various extracellular ligands is well described. Genetically modified mouse models, especially the RAGE knockout (RAGE-KO) mouse, identified the amplification of the immune response as an important function of RAGE. Pro-inflammatory ligands of RAGE are also methylglyoxal-derived advanced glycation end-products, which depend in their quantity, at least in part, on the activity of the methylglyoxaldetoxifying enzyme glyoxalase-1 (Glo1). Therefore, we studied the potential interaction of RAGE and Glo1 by use

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of RAGE-KO mice.

Methods

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Various tissues (lung, liver, kidney, heart, spleen, and brain) and blood cells from RAGE-KO and wildtype mice

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were analyzed for Glo1 expression and activity by biochemical assays and the Glo1 gene status by PCR techniques.

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Results

We identified an about two-fold up-regulation of Glo1 expression and activity in all tissues of RAGE-KO mice. This was result of a copy number variation of the Glo1 gene on mouse chromosome 17. In liver tissue and blood cells,

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the Glo1 expression and activity was additionally influenced by sex with higher values for male than female

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animals. As the genomic region containing Glo1 also contains the full-length sequence of another gene, namely

Conclusion

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Dnahc8, both genes were duplicated in RAGE-KO mice.

A genetic variance in RAGE-KO mice falsely suggests an interaction of RAGE and Glo1 function.

General significance RAGE-independent up-regulation of Glo1 in RAGE-KO mice might be as another explanation for, at least some, effects attributed to RAGE before.

Key words: Advanced glycation end-products; Copy number variation; Dnahc8; Glo1; Receptor for advanced glycation end-products; Sex

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Journal Pre-proof Introduction The receptor for advanced glycation end-products (RAGE) is member of the immunoglobulin superfamily and fulfills multiple functions. As a cell surface molecule, RAGE induces cell signaling pathways leading to inflammation and enhances the adhesion of cells to extracellular matrix and adjacent cells [1, 2], whereas nuclear RAGE mediates recognition and repair of DNA damages [3]. The best studied function of RAGE is the amplification of inflammatory responses [1]. In this role, RAGE acts as pattern recognition receptor of various endogenous molecules released from the cells due to acute or chronic inflammation and physiological stress [4]. To date, advanced glycation end-products (AGEs) [5, 6], S100 proteins [7], high mobility group box 1 protein [8],

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amyloid-β [9], complement component C1q [10], nucleic acids [11] and phospholipids [12] have been identified as

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interaction partners of RAGE.

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The expression of RAGE is induced at inflammatory sites in most tissues , while the lung tissue constitutively expresses RAGE at high level [13]. In lung, RAGE is mainly located at the basolateral site of alveolar type I (AT I)

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cells [14] through which gases exchanges between alveolar air and the blood in the pulmonary capillaries. The

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interaction of RAGE with defined extracellular matrix molecules [15] and RAGE itself [16] seems to be an essential part of its AT I cell-related function contributing to cell spreading [15, 17] and impermeability of the alveolar-

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capillary membrane for proteins and cells [18, 19]. During inflammation, RAGE interacts with 2-integrins and

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supports the trans-endothelial migration of immune cells [20, 21]. Apart from the physiological role of RAGE in AT I cells of the lung tissue, the induced expression of RAGE in

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other cell types of lung and other organs contributes to the pathogenesis of many inflammatory diseases including metabolic disorders (diabetes, obesity), vascular complications, bacterial infections (pneumonia, sepsis), liver cirrhosis, neurodegenerative disorders, asthma and cancer, as well as disorders following physiological stress (ischemia-reperfusion, hypoxia, hyperoxia) [1, 2]. Most studies stressing the importance of RAGE in inflammation have been performed in mice deficient in RAGE [22-30] and mice overexpressing a dominant-negative variant of RAGE (DN-RAGE, without cytoplasmic signaling domain) [31-34] or a soluble variant of RAGE (sRAGE, without transmembrane and cytoplasmic signaling domain) [35, 36]. sRAGE variants also naturally exist due to alternative splicing events [37, 38] or proteolytic cleavage of the membrane-bound receptor by metalloproteinases [39]. Moreover, mice overexpressing full-length RAGE [40-43] and mice treated with recombinant sRAGE [44], RAGEblocking antibody [34] or RAGE-blocking agent (FPS-ZM1 [45]) contributed to the understanding of RAGE function. Among the variety of genetic mouse models different in their RAGE expression, RAGE knockout (RAGEKO) mice are mostly used, especially the RAGE-KO mouse strain generated at the Heidelberg University (RAGE-

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Journal Pre-proof KOHU ) [30]. Latest literature search in the PubMed database [46] revealed more than 150 studies using this mouse strain followed by about 25 studies using the RAGE-KO mouse generated at the Kanazawa University [22]. Other RAGE knockout or transgene mouse strains have been far less frequently used in animal experimental studies (see list of studies using RAGE deficient mouse models in Supplemental Table S1). Initially, research on RAGE began with its identification as a binding molecule of AGEs [5, 6], to be exact Ncarboxymethyllysine (CML) [47] and methylglyoxal-derived hydroimidazolone 1 (MG-H1) [48]. CML and MG-H1 mainly result from the non-enzymatic degradation of fructosyl-lysine residues and reaction of the dicarbonyl compound methylglyoxal with arginine residues of proteins, respectively. Methylglyoxal is a toxic side-product of

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glycolysis and other metabolic pathways. In order to keep the cellular level of methylglyoxal low, it is detoxified by

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the glyoxalase system consisting of glyoxalase-1 (Glo1) as rate-limiting enzyme, glyoxalase-2 and cofactors [49, 50]. Many mouse experimental studies described the RAGE dependency of the pro-inflammatory action of AGEs in

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various organs including heart [51-53], kidney [54-56], brain [24, 57], and also adipose tissue [58]. If this RAGE-

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dependent AGE action is directly caused by AGE-RAGE interaction is still unclear. Moreover, the presence of RAGE itself seems to increase the endogenous formation of methylglyoxal and methylglyoxal-derived AGEs via

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down-regulation of the Glo1 expression as indicated by comparative analysis of wildtype and RAGE-KOHU mice [59-61].

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The RAGE-mediated Glo1 reduction may essentially contribute to the pathophysiological effect of the AGE-RAGE

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stress in chronic inflammatory diseases [62-64]. However, it is rather unclear why in physiological conditions other

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organs than lung (kidney, nerves and skin [59]; liver [60]; skeletal muscle [61]) show a significant RAGE dependency of the Glo1 expression level. Therefore, we hypothesized an indirect effect of RAGE on the Glo1 expression. Comparative Glo1 analyses were performed in lung and other tissues of the RAGE-KOHU mouse strain, and the molecular background responsible for the Glo1 regulation was identified in these mice.

Material and methods Animals We used wildtype (WT) mice, RAGE knockout mice (RAGE-KOHU ; provided by Angelika Bierhaus and Peter P. Nawroth, Department of Internal Medicine, Heidelberg University, Heidelberg, Germany) [30] and cardiomyocyte RAGE transgenic mice (RAGE-TR; provided by Thomas Braun, Department of Cardiac Development and Remodeling, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany ) of the C57BL/6N strain.

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Journal Pre-proof RAGE-KOHU mice overexpress the enhanced green fluorescent protein (EGFP) in all cells [30], whereas RAGE-TR mice specifically overexpress EGFP in cardiomyocytes. Backcrossing of the RAGE-KOHU mice onto C57BL/6N genetic background had already been performed for five generations at the animal facility of the Heidelberg University as well as later for at least 10 generations in our animal facility. Tissues obtained from the mice were either fixed in 4 % phosphate-buffered formaldehyde with subsequent embedding in paraffin or frozen in liquid nitrogen for further analysis. Bronchoalveolar lavage fluid and plasma of heparinized blood were prepared and analyzed for sRAGE with the mouse RAGE DuoSet kit containing mouse recombinant RAGE as standard (R&D Systems; Minneapolis, MN).

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RAGE transfection

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Three human lung cell lines (BEAS-2B, NCI-H358, A549; all cells from the ATCC cell bank; Manassas, VA) were

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used for transfection with the full-length human RAGE cDNA cloned in a pIRES2-EGFP vector (Clontech; Palo Alto, CA) or the empty pIRES2-EGFP vector [17]. NCI-H358 and A549 were cultured in Dulbecco's modified

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Eagle’s medium (Thermo Fisher Scientific, Rockford, IL) supplemented with 10 % fetal calf serum (FCS; Perbio, Bonn, Germany), whereas BEAS-2B cells were cultured in Airway Epithelial Cell Growth medium (Promocell,

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Heidelberg, Germany). Viromer® Red (Lipocalyx, Halle (Saale), Germany) was used as transfection reagent.

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Expression analyses

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Total RNA was isolated by use of the TRIzol™ Reagent (Thermo Fisher Scientific) and cleaned-up using the InviTrap Spin Cell RNA Mini kit (Stratec, Berlin, Germany). For mRNA quantification by gene array, biotin -

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labeled cRNA was synthesized from total RNA by use of the Ambion ® WT Expression kit (Applied Biosystems, Carlsbad, CA) and then subjected to the GeneChip hybridization procedure using GeneChip Mouse 2.0 arrays and GeneChip Fluidics station 450 from Affymetrix® (Santa Clara, CA). All arrays were scanned using the Affymetrix GeneChip Scanner 7G with GeneChip Command Console 3.1 software. Data calculation was performed with the Robin software [65]. For mRNA quantification by PCR, cDNA was synthesized from total RNA by reverse transcription and then amplified with human-specific primers for Glo1 (NM_006708.2; sense: 5`-cagcagaccatgcta cgagtg-3`, anti-sense: 5`-tgactcccagttcttcaaacc-3`) and -actin (NM_001101.4; sense: 5`-gaagtgtgacgtggacatccg -3`, anti-sense: 5`-agcatttgcggtggacgat-3`) or mouse-specific primers for Dnahc8 (NM_013811; sense: 5`-cagatgttcggc aggctggata-3`, anti-sense: 5`-ctcaatccagatggcatccacg-3`) using the GoTaq ® pPCR Mastermix (Promega) in the CFX Connect™ Real-Time System (Bio-Rad, Hercules, CA). Data calculation was performed per -actin (2-CP method) or external cDNA standards as appropriate [66].

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Journal Pre-proof Protein lysates of the tissues were prepared in 1 % phosphate-buffered sodium dodecyl sulfate solution supplemented with protease inhibitors. Equal protein amounts were separated by polyacrylamide gel electrophoresis with subsequent immunoblot procedure using polyclonal rabbit antibodies against RAGE (Abcam, Cambridge, UK), Glo1 (R&D Systems) and GAPDH (Santa Cruz, Santa Cruz, CA). Signal intensities were quantified using the LAS 3000 computer-based imaging system (FUJI Film, Tokyo, Japan) equipped with AIDA 2.0 software (Raytest, Straubenhardt, Germany). Total protein stain of the blotted proteins with 0.5 % amido black solution was used for data normalization. For Glo1 immunohistochemistry, paraffin sections were cut (5 µm), dewaxed, rehydrated and blocked with 5 %

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normal goat serum. Glo1 was stained by use of a monoclonal anti-Glo1 antibody (clone 2F7, 1:300 dilution; BioMac

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GmbH, Leipzig, Germany) as described previously [67]. Images of the stained sections were taken with the

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Axiophot microscope equipped with Spot Camera (Carl Zeiss, Jena, Germany). Gene analyses

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gDNA was isolated from mouse tissues by use of the Quick-DNA TM Microprep kit (Zymo Research, ‎Irvine, CA‎‎ )

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‎ dna then subjected to qPCR amplification with the GoTaq ® qPCR Mastermix (Promega) and mouse-specific primers in the CFX Connect™ Real-Time System (Rio-Rad). We used primer sets for Glo1 (NM_025374.3; sense: 5`-

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ccctgctatgaagttctcgctc-3`, anti-sense: 5`-gagctcaagggtggcttttct-3`), Dnahc8 (NM_013811; sense: 5`-cagatgttcggcag gctggata-3`, anti-sense: 5`-ctcaatccagatggcatccacg-3`) and Npsr1 (NM_175678.3; sense: 5`-cagctgctgccccggctaac-

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3`, anti-sense: 5`-ggttggctggcatggctcagg-3`). Data calculations were performed per Npsr1 as single copy gene (2-CP

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method) [66]. Genomic duplication of the Glo1-/Dnahc8-containing region was identified by another primer set (sense (Dup1_F11): 5`-tagaatgtgttagggaacccactgc-3`, anti-sense (Dup1_R3): 5`-ggccaggtcaacacattactcccag-3` [68]) using the PrimeSTAR® GXL DNA polymerase (Takara, Kusatsu, Japan) that amplifies the duplication transition region. Standard PCR procedures using primers for EGFP (sense: 5`-gacgtaaacggccacaagttcagc-3`, anti-sense: 5`ttgggtctttgctcagggc-3`) and RAGE (sense: 5`-ctgaactcacagccagtgtccc-3`, anti-sense: 5`-ccctgactcggagttggatag-3`) proved the RAGE mouse genotype. Glo1 activity Mechanically lysed tissue samples and whole blood cells were adjusted to the same protein amount in phosphatebuffered saline and transferred to the Glo1 activity assay [69]. In this assay, hemithioacetal was pre-formed by incubating 2 mM glutathione and 2 mM methylgyloxal in 50 mM sodium phosphate buffer (pH 6.6) at 37°C for 10 min and then added to the samples in a UV transparent microplate. S. cerevisiae Glo1 (Sigma-Aldrich, Taufkirchen, Germany) was used as positive control and phosphate-buffered saline as negative control. The Glo1-mediated

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Journal Pre-proof formation of S-D-lactoylglutathione was detected at 240 nm and 37°C for 10 min. Glo1 activity was calculated according to the formula: (absorption/min  reaction volume  sample dilution) / ( 2.86 mM −1 ·cm−1  sample volume) and then normalized per protein amount. Statistics Statistical calculations and data presentations were performed by use of the SigmaStat 3.5 and SigmaPlot 10 software (Systat Software Inc., San Jose, CA). The Student’s t-test (parametric data) or the Rank sum test (nonparametric data) was used for comparing data from RAGE-KOHU and WT mice, whereas the ANOVA test was used

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for multiple comparisons. Data are presented as mean  SD, and P values ≤ 0.05 indicate significant changes.

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Results

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Glo1 expression and activity

RAGE protein is mainly expressed in lung, whereas the protein expression of Glo1 was detectable in all selected

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mouse organs with highest amount in liver (Fig. 1 A). Detailed investigations of the lung tissues showed that the mRNA- and protein amount of Glo1 was up-regulated in RAGE-KOHU mice (Fig. 1B). Though RAGE is marker of

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AT I cells in lung [14], subsequent immunohistochemical analyses of lung tissues did not only indicate RAGE-

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dependent differences of the Glo1 amount in the alveolar areas but also in the airways (Fig. 1C). Moreover, the overexpression of RAGE in lung epithelial cells did not alter the Glo1 mRNA amount as expected (Fig. 1D). For

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that reason, we analyzed different organs as well as whole blood cells of a small number of mice for the RAGE dependency of Glo1 by an activity assay. These analyses indicated that the Glo1 activity was up-regulated in all samples of RAGE-KOHU mice (Fig. 2A). Glo1 up-regulation was significant in all organs investigated with higher values for kidney, lung and heart and lower values for liver and blood cells (Fig. 2A). Further analyses of a large number of mice showed that the Glo1 activity was additionally influenced by sex, which has been identified for liver and blood cells but not for kidney (Fig. 2B). Immunoblot analyses of the identical liver samples indicated that the sex-dependent differences in Glo1 activity correlate with the protein amount (Fig. 2C). Lung tissues did also not show an influence of sex on Glo1 protein amount (Fig. 2C). Reasons for the Glo1 regulation The tissue-independent Glo1 up-regulation in RAGE-KOHU mice could result from the overexpression of EGFP in all cells. However, an influence of EGFP on Glo1 seems unlikely because heart samples obtained from RAGE-TR

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Journal Pre-proof mice overexpressing EGFP in cardiomyocytes did not show higher Glo1 expression levels than WT hearts, whereas heart samples obtained from RAGE-KOHU did (Fig. S1A-B). Another possibility explaining the tissue-independent Glo1 up-regulation in RAGE-KOHU mice could be the absence of the soluble variant of RAGE in body fluids including blood plasma and extracellular spaces including lung lumen (Fig. S1C). However, in this case we still had expected stronger differences in the Glo1 up-regulation between the different types of tissue. We then thought about the potential existence of a Glo1 copy number variance (CNV) reported in mice [68] because the RAGE gene (also called AGER) and the Glo1 gene are both located on chromosome 17 (Fig. 3A). The Glo1 CNV results from the doubling of a genomic region containing Glo1 (Fig. 3A) and has been identified in some

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inbred mouse strains, such as DBA/2, to be the reason for strain-dependent differences in the Glo1 expression [68].

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Therefore, we proved the copy number of Glo1 in mouse gDNA samples obtained from WT and RAGE-KOHU mice of the C57BL/6N strain as well as WT mice of the DBA/2 strain (positive control). Indeed, qPCR analyses of gDNA

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isolated from heart indicated the doubling of the Glo1 gene in RAGE-KOHU mice (Fig. 3B). qPCR analyses of

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gDNA isolated from other tissues confirmed this observation (Fig. S2A). In order to identify whether the identical genomic variation reported before (Fig. 3A) exists in RAGE-KOHU mice, we additionally investigated the copy

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number of the Dnahc8 gene, which is also located in this region, as well as the existence of the duplication transition region. qPCR analyses showed the doubling of the Dnahc8 gene (Fig. 3C, Fig. S2B) and long-range PCR analyses

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the existence of the duplication transition region (Fig. 3D) in RAGE-KOHU mice. As the Dnahc8 CNV could

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mediate effects in RAGE-KOHU mice, we also studied the Dnahc8 mRNA amount in various organs of the C57BL/6N strain. qPCR analyses identified the highest expression of Dnahc8 in testis (Fig. 3E).

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Glo1 doubling and activity

The Glo1/Dnahc8 CNV in RAGE-KOHU mice does not seem to be eliminated by backcrossing to the WT genome due to the relative proximity of the RAGE region knocked out and the genomic region containing Glo1 and Dnahc8 (Fig. 3A). Nevertheless, we identified two male RAGE-KOHU mice having lost the Glo1/Dnahc8 genetic variation after backcrossing procedure (Fig. 4A-B). Both RAGE-KOHU mice showed lower Glo1 activities in tissues and blood cells compared to RAGE-KOHU mice carrying two Glo1 genes (Fig. 4C).

Discussion The present study demonstrated a Glo1 CNV as main reason for the increased Glo1 expression and, therefore, increased Glo1 activity in all tissues of the RAGE-KOHU mouse strain. In this regard, we identified the duplication

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Journal Pre-proof of a genomic region on mouse chromosome 17 containing Glo1. Independent of the genetic variance, male sex was associated with an increased expression and activity of Glo1 in some but not all mouse tissues.

Origin of the Glo1 CNV in RAGE-KOHU mice In 2009, Williams et al. reported about a 475 kb tandem duplication on mouse chromosome 17 (30.18 to 30.65 Mb) that includes full-length Glo1, and also full-length Dnahc8 as well as parts of Btbd9 and Glp1r [68]. The duplication of Glo1 causes an increased Glo1 expression because Glo1 including promoter region exists twice [68]. Of the 71 inbred mouse strains tested, 23 strains including DBA/2 were positive for this tandem duplication. Mice of

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the C57/BL6 background showed no duplication of Glo1-containing region [68] as confirmed by our analysis. The genetic background of the RAGE-KOHU mouse is a mixed background consisting of the C57BL/6N and 129SvEv

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mouse genome with significantly higher amount of the C57BL/6N genome as the result of backcrossing procedures [30]. Early studies on this RAGE-KOHU mouse strain still used B6129 hybrid mice as control cohort [44]. As

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backcrossing procedures over many generations will not completely remove the 129SvEv genome and RAGE is

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located on the same chromosome only 3.16 Mb up-stream of the Glo1-containing duplication region, it is well possible that the Glo1 CNV in RAGE-KO mice originates from the 129SvEv embryonic stem cells used for

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generating the RAGE-KOHU mouse.

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Embryonal stem cells derived from the mouse strain 129 are a common tool for the genetic modification of mice. Several substrains of 129 mice exist which are broadly classified as parental (P), steel (S), Ter (T) or genetically

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contaminated (X) substrain [70]. Williams et al. (2009) tested two of them and identified the duplication of the Glo1-containing region in 129S1/SvImJ mice of the S substrain but not in 129X1/SvJ mice of the X substrain [68]. As 129SvEV mice belong to the S substrain [70] it is, therefore, well possible that the Glo1 CNV in our RAGE-KO mice originates from the genome of the 129SvEv embryonal stem cells. The Glo1-containing duplication region on mouse chromosome 17 is not present in all inbred strains, in part, due to its instability and reversal of the duplicated to the non-duplicated state [68]. This reversal is also suggested for 129X1/SvJ mice [68] of which the embryonal stem cells have been used for the generation of the 52-kDa FK506 binding protein (FKBP52) knock out mouse [71]. FKBP52 is located 1.78 Mb down-stream of Glo1 on mouse chromosome 17, and the FKBP52 knockout mouse has also been tested positive for the Glo1 duplication despite backcrossing to C57BL/6 strain [72]. Assumed that the designation of the 129 mouse substrain (129SvJ which is now designated as 129X1/SvJ [70]) used for the generation of the FKBP52 knock out mouse is correct [72], it

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Journal Pre-proof suggests that genetic manipulations on mouse chromosome 17 could also induce the duplication of the 475 kb region containing Glo1. In each case, the relative proximity of FKBP52, and also RAGE, to the Glo1-containing duplication region seems to prevent the deletion of the Glo1 CNV through the backcrossing procedure. This special problem became also obvious in Glo1 knockout mice described by Shafie et al. (2016) [73]. These mutant mice did not show the expected Glo1 down-regulation in tissues due to a simultaneous Glo1 CNV [73].

Physiological importance of the Glo1 CNV in RAGE-KO mice Lopez-Diez et al. (2017) recently found that experimental diabetes impairs muscle inflammation, neoangiogenesis

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and blood flow recovery after hind limb ischemia which was less pronounced in RAGE-KOHU mice as well as in Glo1 transgenic (Glo1-TR) mice [61]. Our data on RAGE-KOHU mice carrying the Glo1 CNV points to the

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possibility that the above mentioned effects might exclusively be mediated by Glo1. The activation of the AGERAGE axis has been discussed as an important pathway after skeletal muscle ischemia because CML increases in

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the ischemic muscles of WT mice to much higher extent than in Glo1-TR mice [61]. Despite the lack of further

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comparative analyses of RAGE-KOHU mice carrying the Glo1 CNV and Glo1-TR mice, it indicates that RAGE may not be responsible for all cellular effects reported in the past. In particular, this will be the case for in vivo conditions

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associated with the formation of methylglyoxal-derived AGEs. Physiological stress due to ischemia seems to be one

ischemic conditions [74-77].

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of such in vivo conditions because other studies also identified a beneficial effect of Glo1 overexpression in

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Despite the clear disadvantage of the RAGE-KOHU mouse for AGE research, the Glo1 CNV in these mice will not query all cellular effects attributed to RAGE. Many mouse experimental studies have been performed with other RAGE-deficient mouse models possibly not carrying the Glo1 CNV as well as the simultaneous treatment of the mice with recombinant sRAGE scavenging RAGE ligands or monoclonal antibodies blocking the extracellular domain of RAGE (see Supplemental Table S1). In particular, the cell adhesive function of RAGE will be correct [15, 17, 20, 21] which exclusively depends on the extracellular function of RAGE [17]. The RAGE-KOHU mouse strain is widely used in the field of RAGE research for more than 15 years (see Supplemental Table S1). Although it also includes investigations of the AGE-RAGE axis [62-64], the increased expression of Glo1 in this mouse strain is described in few cases only [59-61]. One reason for this discrepancy might be the failure of showing a RAGE-dependent regulation of Glo1 by additional in vitro experiments including cell transfections. Another reason might be the less obvious Glo1 up-regulation in some organs obtained from a mixed mouse population of males and females because of the sex dependency of the Glo1 expression, especially in

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Journal Pre-proof liver. Sex-dependent differences in the cell metabolism and, therefore, methylglyoxal generation may possibly cause the different Glo1 status between males and females. The tissue-dependent influence of sex on the Glo1 expression and activity is also not yet described in the literature and, therefore, unknown for the researchers. In addition to Glo1 CNV, RAGE knockout mice are positive for Dnahc8 CNV because this gene is also located in the 475 kb tandem duplication on chromosome 17 as well. Dnahc8 codes for an axonemal -type dynein heavy chain protein. In physiological conditions, Dnahc8 is highly expressed in testis [78] as also shown by our expression analysis of different mouse tissues. Dnahc8 seems to play a role in the mid to late spermatogenesis [78] but spermatocyte-independent functions of Dnahc8 are not yet described. In this regard, it is also unknown if Dnahc8

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can be induced in other tissues in response to pathophysiological conditions. Therefore, the possible importance of

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the Dnahc8 CNV in RAGE-KO mice is hardly to assess.

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It is possible to eliminate the duplication region containing Glo1 and Dnahc8 in RAGE-KOHU mice by backcrossing procedures in order to specify the physiological importance of RAGE? Yes, it is as suggested by our analysis.

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However, the elimination of the Glo1/Dnahc8 CNV will be a rare event. Therefore, RAGE-KOHU mice carrying no

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Glo1/Dnahc8 CNV have to be selected specifically. Comparative studies of RAGE-KOHU mice with and without Glo1 up-regulation would have the potential to investigate the combined role of RAGE and Glo1 in pathological situations including ischemia. Thus, RAGE-KOHU will still be an interesting tool in the future experimental research.

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available [25].

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In case of more precise RAGE-dependent animal studies , CRISPR-Cas9-generated RAGE-KO mice are now

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In conclusion, our findings confirm the hypothesis that others than direct RAGE-related effects are responsible for the Glo1 up-regulation in the RAGE-KOHU mouse strain. The Glo1 up-regulation due to Glo1 gene doubling identified in these RAGE-KOHU mice will be responsible for some but not all effects attributed to RAGE before.

Acknowledgements This project was supported by grant of the Deutsche Forschungsgemeinschaft (BA2077/4-2). We thank Anika Küttner and Carsten Schulz for technical assistance.

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Journal Pre-proof Figure legends Fig.1. Immunoblot detection of RAGE and Glo1 protein in selected mouse organs (A). Glo1 mRNA and protein amount in lung tissues of WT and RAGE-KOHU mice as determined by gene microarray and immunoblot, respectively (B). RAGE-dependent detection of Glo1 in mouse lung sections (airways are marked by arrows) by immunohistochemistry (C). Glo1 mRNA amount 48 h after transfection of human immortal bronchial epithelial cells (BEAS-2B) and alveolar epithelial cell-derived tumor cell lines (NCI-H358, A549) with human RAGE cDNA cloned in an EGFP expression vector (D). In B, data are means  SD (n = 12 each genotype) with

***

P ≤ 0.001 vs.

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successful RAGE transfection by EGFP positivity of the cells.

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WT. In D, data are means  SD (n = 3) with * P ≤ 0.05 vs. control transfection. Fluorescent microscopy identified the

Fig. 2.

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Enzymatic assay quantified the Glo1 activity in selected mouse organs and whole blood cells (mainly erythrocytes)

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of WT and RAGE-KOHU mice independent of sex (A) and dependent on sex (B). Sex-dependent Glo1 protein amount in mouse liver and lung as determined by immunoblot detection (C). Data are means  SD (n = 3 female

Fig. 3.

†††

P ≤ 0.001 vs. same sex and genotype.

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P ≤ 0.01,

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mice each genotype in A, n = 15 each genotype and sex in B and C) with * P ≤ 0.05, ** P ≤ 0.01 vs. WT and † P ≤ 0.05,

Scheme of the genomic arrangement of Glo1 and RAGE on mouse chromosome 17 without (upper) and with (lower) doubling of a 475 kb region containing Glo1 and Dnahc8 as well as parts of Btbd9 and Glp1r (A). In A, the positions of the genes on chromosome 17 are derived from the mouse genome assembly version MGSCv39 as also used by Williams et al. (2009) [68]. Quantification of Glo1 (B) and Dnahc8 (C) per single copy gene (Npsr1) by real-time qPCR using specific primer sets and mouse gDNA isolated from heart samples of C57BL/6N (WT, RAGE-KOHU ) and DBA/2 (WT) mice. Agarose gel electrophoresis of the long-range PCR using a primer set that amplifies a gDNA fragment (4.5 kb) across the boundary of the Glo1-/Dnahc8-containing duplication region (D). mRNA amount of Dnahc8 in selected mouse tissues of C57BL/6N WT mice (E). In B-C, data are means  SD (n = 5 each genotype and strain) with ** P ≤ 0.01 and *** P ≤ 0.001 vs. WT. In E, data are means  SD (n = 4 each tissue).

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Journal Pre-proof Fig. 4. Identification of two RAGE-KOHU mice without Glo1 CNV as shown by the absence of the gDNA amplicon (4.5 kb) representing the boundary of the Glo1-/Dnahc8-containing duplication region (A) and the normal amount of Glo1 and Dnahc8 in the gDNA isolated from heart (B). Glo1 activity in tissue or blood cell lysates derived from both RAGE-KOHU mice without Glo1 CNV compared to RAGE-KOHU mice with Glo1 CNV. In A, C57BL/6N WT

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mice were used as negative control and DBA/2 WT mice as positive control.

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Journal Pre-proof Figure legends of supplemental data Fig. S1. EGFP-dependent Glo1 protein amount in heart samples of different RAGE genotypes as determined by immunoblot (A, B). Quantification of the soluble RAGE protein by ELISA in blood plasma and lung lavage obtained from WT and RAGE-KOHU mice (C). Data are means  SD (n = 8 (4 females, 4 males) each genotype).

Fig. S2. Quantification of Glo1 (B) and Dnahc8 (C) per single copy gene (Npsr1) by real-time qPCR using specific primer

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sets and mouse gDNA isolated from liver and kidney samples of WT and RAGE-KOHU mice. Data are means  SD

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(n = 5 each genotype) with *** P ≤ 0.001 vs. WT.

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Journal Pre-proof Highlights RAGE interacts with methylglyoxal-derived AGEs, which are reduced by Glo1 activity.

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Glo1 expression and activity are up-regulated in tissues of RAGE knockout mice.

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Glo1 up-regulation is not caused by RAGE deficiency but Glo1 copy number variation.

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Glo1 expression and activity in some tissues are additionally increased in males.

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Effects attributed to RAGE could be caused by Glo1 up -regulation in these mice.

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Figure 1

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