RETREG1-mediated reticulophagy is activated by an ATF4-CEBPG/C/EBPγ heterodimer and confers protection against lipotoxicity

Excessive fatty acid triggers endoplasmic reticulum (ER) stress, leading to lipotoxicity, which plays a vital role in the pathogenesis of metabolic dysfunction-associated steatotic liver disease (MASLD). Reticulophagy is recently identified as an integral process in maintaining ER homeostasis during ER stress. However, our knowledge of reticulophagy in lipotoxicity remains limited, and the underlying molecular mechanisms are unclear. Here we showed that mild, short-term lipotoxicity induced by palmitic acid stimulated reticulophagy <i>in vitro</i>, mediated primarily by the selective receptor RETREG1. Knockdown of <i>RETREG1</i> in HepG2 cells and primary hepatocytes exacerbated palmitic acid-induced cell damage and death. Having demonstrated the indispensability of ATF4 and CEBPG/C/EBPγ in transcriptional upregulation of <i>RETREG1</i>, we found that ATF4 forms a heterodimer with CEBPG and identified their binding sites in the promoter and enhancer regions of <i>RETREG1</i> gene. In mice with acute hepatic lipotoxicity, RETREG1-mediated reticulophagy was activated, conferring protection against liver injury, as <i>retreg1</i> knockout mice exhibited more severe liver injury than wild-type mice. In contrast, reticulophagy initiation was defective in a high fat diet-induced mouse model of MASLD, possibly due to decreased gene expression of <i>Retreg1</i> driven by the suppression in ATF4 and CEBPG. Our study underscores the crucial role of RETREG1-mediated reticulophagy, which is co-regulated by ATF4 and CEBPG, in response to lipotoxicity, suggesting that activation of reticulophagy may represent a strategy against MASLD. <b>Abbreviations:</b><i>ATF4</i>/<i>Atf4</i>:activating transcription factor 4;ATL3: atlastin GTPase 3; Baf A1: bafilomycin A₁;CAREs:CEBP-ATF response elements; CASP9:caspase9;<i>CCPG1</i>/<i>Ccpg1</i>:cell cycle progression 1; CEBPB/C/EBPβ: CCAAT enhancer bindingprotein beta; CEBPG/C/EBPγ:CCAAT/enhancerbinding protein gamma; ChIP: chromatin immunoprecipitation; Co-IP:co-immunoprecipitation; CQ: chloroquine; DDIT3: DNA damage inducibletranscript 3; EIF2A: eukaryotic translation initiation factor 2A;EIF2AK3: eukaryotic translation initiation factor 2 alpha kinase 3;ER: endoplasmic reticulum; ERN1: endoplasmic reticulum to nucleussignaling 1; Fa/R: fasted overnight followed by refeeding with ahigh-carbohydrate, fat-free diet; FBS: fetal bovine serum; GOT1/AST:glutamic-oxaloacetic transaminase 1, soluble;GPT/ALT:glutamic pyruvic transaminase, soluble; HCD:high-carbohydrate diet; H&amp;E: hematoxylin and eosin; HFD: high-fatdiet; <i>Hmox1</i>:heme oxygenase 1; IHC: immunohistochemistry;KRT18/CK18: keratin 18; LDH: lactatedehydrogenase; MAP1LC3/LC3: microtubule-associated protein 1 lightchain 3; MASLD: metabolic dysfunction-associated steatotic liverdisease; MDA: malondialdehyde; ND: normalchow diet; <i>Nfe2l2</i>:nuclear factor, erythroid derived 2, like 2; <i>Nqo1</i>:NAD(P)H dehydrogenase, quinone 1; PA: palmitic acid; PCR: polymerasechain reaction; RT-qPCR: quantitativereal-time PCR; <i>RETREG1</i>/<i>FAM134B</i>:reticulophagy regulator 1; <i>RTN3</i>/<i>Rtn3</i>:reticulon 3; <i>SEC62</i>/<i>Sec62</i>:SEC62 homolog, preprotein translocation; <i>Sod2</i>:superoxide dismutase 2, mitochondrial;<i>SQSTM1</i>/<i>Sqstm1</i>:sequestosome 1; <i>TEX264</i>/<i>Tex264</i>:testis expressed 264; TEM: transmission electron microscopy; TG:triglyceride; UPR: unfolded protein response; WT: wild-type; XBP1:X-box binding protein 1