All-trans retinoic acid induces GADD34 gene expression via transcriptional regulation by Six1-TLE3 and post-transcriptional regulation by p38-TTP in skeletal muscle

All-trans retinoic acid (ATRA) increases the sensitivity to unfolded protein response (UPR) in differentiating leukemic blasts. The downstream transcriptional factors of PERK, a major arm of UPR, regulates muscle differentiation. However, the role of growth arrest and DNA damage-inducible protein 34 (GADD34), one of the downstream factors of PERK, and the effects of ATRA on GADD34 expression in muscle remain unclear. In this study, we identified ATRA increased the GADD34 expression independent of the PERK signal in the gastrocnemius muscle of mice. ATRA up-regulated GADD34 expression through the transcriptional activation of it via inhibiting the interaction of homeobox Six1 and transcription co-repressor TLE3 with the MEF3-binding site on the GADD34 gene promoter in myoblasts. ATRA also inhibited the interaction of TTP, which induces mRNA degradation, with AU-rich element on GADD34 mRNA via p38 MAPK, resulting in the instability of GADD34 mRNA. Overexpressed GADD34 in myoblasts changes the type of myosin heavy chain in myotubes. These results suggest ATRA increases GADD34 expression via transcriptional and post-transcriptional regulation in myoblasts, which changes muscle fiber type in myotubes.

GADD34 gene promoters to ATRA using a luciferase assay with undifferentiated C2C12 1 myoblasts and HEK293 cells. The overexpression of RARs increased the luciferase activity of 2 pGADD34-0.5k in both C2C12 myoblasts and HEK293 cells. ATRA increased the luciferase 3 activity of pGADD34-0.5k in C2C12 myoblasts expressing RAR and RAR, but not RAR ( Fig   4   2A). ATRA dose-dependently increased the GADD34 gene promoter activity in C2C12 myoblasts, 5 unlike in HEK293 cells ( Fig 2B). Surprisingly, TTNPB, a major agonist of RAR, did not increase 6 the GADD34 gene promoter activity, although TTNPB increased the mRNA expression of the 7 target gene (Appendix Fig S1A and B) (Thé et al, 1990). Unlike TTNPB, ATRA can activate the 8 non-genomic p38 and ERK MAPK signals via extranuclear RAR/ and RAR, respectively 9 (Tanoury et al, 2013;Bouchard & Paquin, 2013;Khatib et al, 2019). Next, we tested whether 10 these MAPK signals are involved in the regulation of the GADD34 expression by ATRA. As a 11 result, the ATRA-induced GADD34 gene promoter activity was blocked by p38 inhibitor SB203580 12 or ERK inhibitor FR180204 via RAR/ or RAR, respectively (Appendix Fig S1C). Next, to 13 explore the muscle-specific regulatory element of ATRA on the human GADD34 gene promoter, 14 several reporter constructs lacking portions of the 5'-promoter region of the human GADD34 deletion analyses suggest that the sequence from -162 to -131 is responsible for ATRA-1 dependent activation of the human GADD34 gene promoter activity in C2C12 myoblasts ( Fig 2C). To explore the molecular mechanism underlying the responsiveness of the GADD34 gene to 6 ATRA in C2C12 myoblasts, we searched for undifferentiated muscle-specific transcription factor 7 binding sites from -162 to -131 on the human GADD34 gene promoter with Matlnspector-8 Genomatix. A search for transcription factor binding motifs with this region suggested five 9 potential binding sites: three MEF3 sites, Pax3, and Sox6 (Fig3 A). Based on this search, we 10 overexpressed Six1, a major transcription factor for the MEF3-binding site, Pax3, and Sox6 with 11 GADD34 gene promoter constructs in C2C12 myoblasts. Although each transcription factor 12 decreased the luciferase activity of pGADD34-0.16k, Six1 did not affect the activity of pGADD34-and GADD34 in myoblasts. As a result, p38 and ERK inhibitors blocked the decrease of the Six1 1 mRNA expression and the increase of the GADD34 mRNA expression induced by ATRA 2 (Appendix Fig S1D). These results indicated that ATRA increases the GADD34 expression via 3 suppression of the binding of Six1 with the MEF3-2 sequence on the human GADD34 gene 4 promoter by decreasing the expression of Six1. Six1 requires the Eya family as the co-activator for transcriptional activation but requires the TLE 9 family as the co-repressor for transcriptional repression (Jennings & Horowicz, 2008). To define 10 a co-repressor that is responsible for the Six1-mediated reduction of the GADD34 gene 11 expression, we first analyzed the mRNA expression levels of the TLE family (TLE1-6) by qPCR 12 with the absolute standard curve method. Among the TLE family, the TLE1, TLE3, and TLE5 to TLE3-knockdown-but not TLE1-and TLE5-knockdown-pGADD34-0.13k exhibited no 1 increase in response to these knockdowns ( Fig 5C). The mutated promoter constructs shown in 2 Fig 3E revealed that the MEF3-2 sequence is essential for the activation of the GADD34 promoter 3 by TLE3-knockdown ( Fig 5D). TLE3-knockdown blocked the effects of the luciferase activity of 4 pGADD34-0.16k by the overexpression of Six1 or ATRA treatment (Fig 5E). On the other hand, 5 ATRA did not change the mRNA expression of TLE1, TLE3, and TLE5 in C2C12 myoblasts 6 (Appendix Fig S2B). These results indicate that ATRA induces the transcriptional activity of the 7 GADD34 gene through the inhibition of the collaborative work in TLE3 and Six1 through the 8 decreased expression of Six1. 9 10 ATRA increases the stability of GADD34 mRNA via TTP in a p38 dependent manner

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Thus far, we have demonstrated that ATRA increases the GADD34 expression by transcriptional activity of GADD34-3'UTR, the effects of these treatments on its activity in C2C12 cells were 1 canceled by TTP-knockdown using TTP-specific siRNA (Fig 6E; Appendix Fig S3C-E). In addition myoblasts. Likewise, although a recent study reported that GADD34 levels were decreased in 1 satellite cells collected from injured muscle with increased ATF4 (Xiong et al, 2017), the 2 mechanism of this paradoxical result is unknown. Since muscle atrophy can occur with differential 3 sensitivity due to selective skeletal muscle fiber subtypes in various pathological conditions, 4 muscle fiber type change is suggested to be an important factor for muscle wasting (Wanga and 5 Pessinn 2013). Muscle atrophy is a frequent complication in CKD patients (Carrero et al, 2013).

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Interestingly, the plasma levels of vitamin A (retinol or ATRA) increase in CKD patients (Gueguen 7 et al, 2005;Jing et al, 2016). It has been reported that the percentage of type 1 slow fiber in type 8 2 fast fiber increases in CKD mice through a decrease in the expression of MYHC2a (Tamaki et 9 al, 2014). However, the molecular mechanisms underlying the muscle fiber type change in CKD 10 are largely unknown. In the present study, we revealed that the overexpression of GADD34 11 increases the MYHC1 protein expression, which is expressed in type 1 slow fibers, and decreases (whereby the MyoD expression turns extra muscle cells into muscle) is impaired in mouse 1 embryonic fibroblasts in Six1/4 double mutant mice because Six1 and MyoD interact with the 2 Myogenin gene promoter to differentiate muscle (Santolini et al, 2016). On the other hand, TLE3 3 downregulates myogenic differentiation via the suppression of MyoD activity (Kokabu et al, 2017).

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Six1 requires the Eya family as the co-activator for transcriptional activation but the TLE family as 5 the co-repressor for transcriptional suppression. In the present study, we suggested that-among 6 the TLE family-TLE3 is the most abundant gene in myoblasts and is an important co-repressor 7 of Six1 for the regulation of GADD34 gene transcriptional activity, which consequently changes 8 the ratio of muscle fiber type. In addition, we confirmed that other undifferentiated muscle-specific 9 transcriptional factors, Pax3 and Sox6, also suppress the GADD34 gene promoter activity. These 10 findings suggest that the expression of GADD34 may constantly be kept low by not only Six1-11 TLE3 but also by undifferentiated muscle-specific transcriptional factors. However, the reason 12 why the expression of GADD34 is constantly low in myoblasts is unknown.

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The expression of genes is determined by transcriptional regulation and post-transcriptional 14 regulation via mRNA stability. The 3'UTR works as one of the regulatory components of mRNA 15 degradation by promoting or inhibiting the deadenylation via interaction with RBP (Mayr, 2019).
with inflammatory-related genes, a recent study revealed that other target mRNAs are related to 1 cancer, apoptosis, and other conditions (Kaempfer, 2003;Schuster & Hsieh, 2019;Lal et al, 2005).

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However, UPR-related mRNA has not previously been reported as an ARE-dependent target for 3 stabilization. TTP and HuR, the most famous RBP interacted with ARE (5'-AUUUA-3') on the 4 3'UTR, to induce mRNA degradation and stabilization, respectively. However, unlike TTP, HuR 5 did not regulate GADD34 mRNA stability in the present study. The distinction between HuR and 6 TTP binding has been reported to involve subtle content features: TTP: 5'-7 (Bhandare et al, 2017). In 8 other words, HuR strongly prefers U-rich sequences, whereas TTP prefers AU-rich with 9 increasing A content. This may explain why HuR did not interact with the ARE sequences of 10 GADD34 mRNA in the present study. ATRA activates the non-genomic p38 MAPK via 11 extracellular RAR and RAR (Tanoury et al, 2013). Furthermore, phosphorylated p38 inhibits 12 the TTP interaction with ARE on target mRNA (Bhattacharyya et al, 2011). Our results indicated 13 that ATRA stabilizes GADD34 mRNA by inhibiting the interaction between its mRNA and TTP via 14 p38 MAPK. Although it has been reported that ATRA regulates the stabilization of mRNA via 15 interaction between apo-cellular retinoic acid-binding protein 2 (apo-CRABP2) and HuR 16 (Vreeland et al, 2014), this is the first study to report that ATRA-dependent MAPK activation increases-via ARE-the mRNA stability of the 3'UTR of the target mRNA. Because p38 MAPK 1 signaling regulates a large number of cellular processes, the mechanism underlying mRNA 2 stabilization by ATRA may also be involved in many mRNAs other than GADD34 mRNA.

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Although the Six genes, especially Six1, are expressed in multiple organs during mammalian 4 development, their expression in global tissues decreases as the individual grows to adulthood 5 (Kumar, 2009). Six1 also controls muscle physiology not only in embryogenesis but also in the 6 adult (Maire et al, 2020). In soleus, Six1 deficiency reduced MYHC2a fiber in 3-week-old C57BL6 7 mice and caused the complete disappearance of the expression of MYHC2a fiber in 12-week-old 8 C57BL6 mice (Sakakibara et al, 2016). These results imply that Six1 regulates fast-fiber type 9 acquisition and maintenance in adult mice. Although the effects of GADD34 on skeletal muscle in 10 vivo are unknown, our study revealed that GADD34, which is downregulated by Six1, suppressed 11 the MYHC2a expression in myoblasts. Thus, GADD34 may mediate the regulation of the

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Human recombinant TNF was purchased from Invitrogen (Carlsbad, CA, USA). Puromycin was 14 purchased from Funakoshi (Tokyo, Japan). Chemi-Lumi One Super was purchased from Nacalai TOYOBO KOD one™ PCR Master Mix -Blue-was purchased from TOYOBO (Osaka, Japan). 1 RNA ChIP-IT ® was purchased from Active Motif (Tokyo, Japan). Graduate School. Eight-week-old male C57BL/6J mice were purchased from Japan SLC 7 (Shizuoka, Japan) and individually caged in a climate-controlled room (22 ± 2°C) with a 12-h light-8 dark cycle. These mice were randomly divided into two groups and a total of 1 mg/kg body weight 9 of ATRA or 1% DMSO (control) prepared in sterile saline was intraperitoneally administered, then 10 the mice were sacrificed 24 h later. Before sacrifice, mice were anesthetized with a total of 0.1 11 mg/kg body weight of buprenorphine hydrochloride and a total of 50 mg/kg body weight of 12 pentobarbital sodium salt, and tissues were removed. The present study was approved by the 13 Animal Experimentation Committee of Tokushima University School of Medicine (animal ethical 14 clearance No. T30-66) and was carried out in accordance with guidelines for the Animal Care and use Committee of Tokushima University School of Medicine.

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Quantitative PCR 7 Total RNA was isolated from kidney, liver, EA, GM, femur, C2C12 myoblasts, C2C12 myotubes, 8 and HEK293 cells using an QIAzol ® Lysis Reagent. Real-time quantitative PCR assays were 9 performed using an Applied Biosystems StepOne qPCR instrument. In brief, the cDNA was 10 synthesized from 1 g of total RNA using a reverse transcriptase kit (Invitrogen, Carlsbad, CA) 11 with an oligo-dT primer. After cDNA synthesis, quantitative real-time PCR was performed in 5 l 12 of SYBR Green PCR master mix using a real-time PCR system (Applied Biosystems).

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Amplification products were then analyzed by a melting curve, which confirmed the presence of 14 a single PCR product in all reactions (apart from negative controls). The quantification of given 15 genes was expressed as the mRNA level normalized to a ribosomal 18S housekeeping gene 16 using the ΔΔCt method. Quantitative expression values were calculated from an absolute standard curve method using the plasmid template for each target gene. The primer sequences 1 used for real-time PCR analysis are shown in Appendix Table S1.

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Reporter plasmid construction 4 The promoter fragment of luciferase reporter plasmid pGADD34-0.5k generated by Eurofins 5 Japan (Tokyo, Japan) was subcloned into a pGL-3 vector (Promega, Madison, WI, USA) by 6 restriction enzyme cutting site KpnI/HindIII. Luciferase reporter plasmids GADD34-3'UTR was 7 constructed by PCR amplification of human genomic cDNA as a template using gene-specific 8 primers (Appendix Table S2). These products were subcloned into a pGL-3 vector. Deleted 9 reporter plasmid pGADD34-0.3k was cloned by the digestion of pGADD34-0.5k using DpnI/HindIII.

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Prepared nuclear extracts (15 g) were incubated with the radiolabeled probe in binding buffer 7 [10 mM (Tris-HCl), pH7.5, 1 mM DTT, 1 mM EDTA, 10% Glycerol, 1 mM MgCl2, 0.25 mg/ml 8 bovine serum albumin, 2.5 g/ml salmon sperm DNA and 2 g poly(dI-dC)] in a final volume of 9 20 l for 30 min at room temperature. The specificity of the binding reaction was determined with 10 a 100-fold molar excess of the indicated cold competitor oligonucleotide. The reaction mixture 11 was then subjected to electrophoresis on a 5% polyacrylamide gel with 0.25×TBE running buffer 12 for 2 h at 150 V. The gel was dried and analyzed with an image scanner (FLA-9000 Starion, Tokyo, 13 Japan). 1 instructions. Briefly, the C2C12 myoblast cells were cultured to 80% confluence in 10-cm dishes 2 and treated with 1 M ATRA. The C2C12 cells were collected and lysed in lysis buffer. The cell 3 extract was prepared and incubated with RNA ChIP buffer pre-conjugated with TTP antibodies or 4 control mouse IgG at 4°C for 16 h. The complexes were treated with Proteinase K for 1 h at 45°C 5 and 1.5 h at 65°C. Immunoprecipitated RNA in the precipitates was purified using QIAzol ® Lysis 6 reagent and analyzed for GADD34 by RT-qPCR. The myotube diameters were determined as previously reported (Niida et al, 2020). Under a 10 fluorescence microscope (BIOREVO BZ-9000, Keyence), three photographs per cell-culture well 11 were obtained in a high-power field. We measured the diameter at the middle portion of the 12 myotube with the built-in BZ-II analyzer software program. We measured the diameters of 100 13 myotubes/group. 14 15 Surface Sensing of Translation (SUnSET) assay in C2C12 cells C2C12 was incubated with or without 1 M puromycin for 30 min before collecting the cells and 1 washed with PBS as previously described (Lim et al, 2017). Puromycin-labeled proteins were 2 detected by Western blotting, as shown above.

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Statistical analysis 5 Data were collected from more than two independent experiments and were reported as the mean 6 and SEM. The statistical analysis for two-group comparison was performed using a two-tailed 7 Student's t-test, or one-way ANOVA with Tukey-Kramer post-hoc test for multi-group comparison.

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E Western blotting of GADD34 in C2C12 cells myoblasts with the indicated concentrations of 12 ATRA.

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A A schematic illustration of the human GADD34 gene promoter in the upper panels. pGADD34-8 0.5k and pCMV- were transfected with pSG5-RAR (, , ) and pSG5-RXR, or empty vector 9 and incubated in the presence of 1 M ATRA or DMSO (NT) as a vehicle for 24 h in C2C12 and 10 HEK293 cells (n = 3-4).

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Data information: In (A), data are presented as the mean ± SEM. *p < 0.05 vs. empty vector. #p 3 < 0.05 (one-way ANOVA with a Student-Newman post-hoc test). In (B), data are presented as 4 the mean ± SEM. *p < 0.05 vs. NT (two-tailed unpaired Student's t-test). In (C), data are presented 5 as the mean ± SEM. *p < 0.05 (two-tailed unpaired Student's t-test). Similar results were obtained 6 from independent experiments.

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MEF3 sequences: box) in the human GADD34 gene promoter region.

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G EMSAs using 32 P-labelled GADD34-MEF3 as probes. EMSAs were performed with nuclear 14 extracts (N.E.) from C2C12 myoblasts treated with 1 M ATRA or DMSO as a vehicle for 24 h.