CREG1 attenuates doxorubicin-induced cardiotoxicity by inhibiting the ferroptosis of cardiomyocytes.

OBJECTIVE: Doxorubicin (DOX)-induced cardiotoxicity limits the application of DOX in cancer patients. Currently, there is no effective prevention or treatment for DOX-induced cardiotoxicity. The cellular repressor of E1A-stimulated genes (CREG1) is a cardioprotective factor that plays an important role in the maintenance of cardiomyocytes differentiation and homeostasis. However, the role and mechanism of CREG1 in DOX-induced cardiotoxicity has not yet been elucidated. METHODS: In vivo, C57BL/6J mice, CREG1 transgenic and cardiac-specific CREG1 knockout mice were used to establish a DOX-induced cardiotoxicity model. H&E staining, Masson's trichrome, WGA staining, real-time PCR, and western blotting were performed to examine fibrosis and ferroptosis in the myocardium. In vitro, neonatal mouse cardiomyocytes (NMCMs) were cultured and stimulated with DOX, CREG1-overexpressed adenovirus, and small interfering RNA was used to establish CREG1 overexpression or knockdown cardiomyocytes. Transcriptomics, real-time PCR, western blotting, and immunoprecipitation were used to examine the roles and mechanisms of CREG1 in cardiomyocytes ferroptosis. RESULTS: The mRNA and protein levels of CREG1 were reduced in the hearts and NMCMs after DOX treatment. CREG1 overexpression alleviated myocardial damage and inhibited DOX-induced ferroptosis in the myocardium. CREG1 deficiency in the heart aggravated DOX-induced cardiotoxicity and ferroptosis. In vitro, CREG1 overexpression inhibited cardiomyocytes ferroptosis induced by DOX, and CREG1 knockdown aggravated DOX-induced cardiotoxicity. Mechanistically, CREG1 inhibited the mRNA and protein expression of pyruvate dehydrogenase kinase 4 (PDK4) by regulating the F-box and WD repeat domain containing 7 (FBXW7)-forkhead box O1 (FOXO1) pathway. PDK4 deficiency reversed the effects of CREG1 knockdown on cardiomyocytes ferroptosis following DOX treatment. CONCLUSION: CREG1 alleviated DOX-induced cardiotoxicity by inhibiting ferroptosis in cardiomyocytes. Our findings may help clarify the new roles of CREG1 in the development of DOX-induced cardiotoxicity.

Creg1 Regulates Erythroid Development via TGF-ß/Smad2-Klf1 Axis in Zebrafish.

Understanding the regulation of normal erythroid development will help to develop new potential therapeutic strategies for disorders of the erythroid lineage. Cellular repressor of E1A-stimulated genes 1 (CREG1) is a glycoprotein that has been implicated in the regulation of tissue homeostasis. However, its role in erythropoiesis remains largely undefined. In this study, it is found that CREG1 expression increases progressively during erythroid differentiation. In zebrafish, creg1 mRNA is preferentially expressed within the intermediate cell mass (ICM)/peripheral blood island (PBI) region where primitive erythropoiesis occurs. Loss of creg1 leads to anemia caused by defective erythroid differentiation and excessive apoptosis of erythroid progenitors. Mechanistically, creg1 deficiency results in reduced activation of TGF-ß/Smad2 signaling pathway. Treatment with an agonist of the Smad2 pathway (IDE2) could significantly restore the defective erythroid development in creg1-/- mutants. Further, Klf1, identified as a key target gene downstream of the TGF-ß/Smad2 signaling pathway, is involved in creg1 deficiency-induced aberrant erythropoiesis. Thus, this study reveals a previously unrecognized role for Creg1 as a critical regulator of erythropoiesis, mediated at least in part by the TGF-ß/Smad2-Klf1 axis. This finding may contribute to the understanding of normal erythropoiesis and the pathogenesis of erythroid disorders.

CREG1 promotes bovine placental trophoblast cells exosome release by targeting IGF2R and participates in regulating organoid differentiation via exosomes transport.

BACKGROUND: Placental exosomes are a kind of intercellular communication media secreted by placental cells during pregnancy, exosomogenesis and release are regulated by many secretory glycoproteins. CREG1 is a kind of secreted glycoprotein widely expressed in various organs and tissues of the body, which inhibits cell proliferation and enhances cell differentiation. The aim of this study was to explore the role of CREG1 in regulating exosomogenesis during the proliferation and differentiation of placental trophoblast cells in early pregnant dairy cows by targeting IGF2R and participating in regulating organoid differentiation via exosomes transport. METHODS: Molecular biological methods were firstly used to investigate the expression patterns of CREG1, IGF2R and exosomal marker proteins in early placental development of pregnant dairy cows. Subsequently, the effects of CREG1 on the formation and release of bovine placental trophoblast (BTCs) derived exosomes by targeting IGF2R were investigated. Further, the effects of CREG1 on the change of gene expression patterns along with the transport of exosomes to recipient cells and participate in regulating the differentiation of organoids were explored. RESULTS: The expression of CREG1, IGF2R and exosomal marker proteins increased with the increase of pregnancy months during the early evolution of placental trophoblast cells in dairy cows. Overexpression of Creg1 enhanced the genesis and release of exosomes derived from BTCs, while knocking down the expression of Igf2r gene not only inhibited the genesis of exosomes, but also inhibited the genesis and release of exosomes induced by overexpression of CREG1 protein. Interestingly, IGF2R can regulate the expression of CREG1 through reverse secretion. What's more, the occurrence and release of trophoblast-derived exosomes are regulated by CREG1 binding to IGF2R, which subsequently binds to Rab11. CREG1 can not only promote the formation and release of exosomes in donor cells, but also regulate the change of gene expression patterns along with the transport of exosomes to recipient cells and participate in regulating the early development of placenta. CONCLUSIONS: Our study confirmed that CREG1 is involved in the exosomogenesis and release of exosomes during the proliferation and differentiation of placental trophoblast cells in early pregnant dairy cows by targeting IGF2R, and is involved in the regulation of organoid differentiation through exosome transport.

CREG1 deficiency impaired myoblast differentiation and skeletal muscle regeneration.

BACKGROUND: CREG1 (cellular repressor of E1A-stimulated genes 1) is a protein involved in cellular differentiation and homeostasis regulation. However, its role in skeletal muscle satellite cells differentiation and muscle regeneration is poorly understood. This study aimed to investigate the role of CREG1 in myogenesis and muscle regeneration. METHODS: RNA sequencing data (GSE8479) was analysed from the Gene Expression Omnibus database (GEO, https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi). We generated Creg1 knockdown and skeletal muscle satellite cells specific Creg1 overexpression mice mediated by adeno-associated virus serotype 9 (AAV9), skeletal muscle mature myofibre Creg1 knockout mice (myoblast/Creg1MKO), and control mice Creg1flox/flox (Creg1fl/fl) as in vivo models. The mice were injected into tibialis anterior (TA) muscle with 100 µL of 10 µM cardiotoxin to establish a muscle regeneration model. Creg1fl/fl and Creg1MKO mice were treated with AAV-sh-C-Cbl (2 × 1010 genomic copies/mouse) to silence C-Cbl in the TA muscle. 293T and C2C12 cells were transfected with plasmids using lipofectamine RNAi MAX in vitro. Mass spectrometry analyses and RNA sequencing transcriptomic assay were performed. RESULTS: We analysed the transcriptional profiles of the skeletal muscle biopsies from healthy older (N = 25) and younger (N = 26) adult men and women in GSE8479 database, and the results showed that Creg1 was associated with human sarcopenia. We found that Creg1 knockdown mice regenerated less newly formed fibres in response to cardiotoxin injection (~30% reduction, P < 0.01); however, muscle satellite cells specific Creg1 overexpression mice regenerated more newly formed fibres (~20% increase, P < 0.05). AMPKa1 is known as a key mediator in the muscle regeneration process. Our results revealed that CREG1 deficiency inhibited AMPKa1 signalling through C-CBL E3-ubiquitin ligase-mediated AMPKa1 degradation (P < 0.01). C-CBL-mediated AMPKa1 ubiquitination was attributed to the K48-linked polyubiquitination of AMPKa1 at K396 and that the modification played an important role in the regulation of AMPKa1 protein stability. We also found that Creg1MKO mice regenerated less newly formed fibres compared with Creg1fl/fl mice (~30% reduction, P < 0.01). RNA-seq analysis showed that CREG1 deletion in impaired muscles led to the upregulation of inflammation and DKK3 expression. The TA muscles of Creg1MKO mice were injected with AAV-vector or AAV-shC-Cbl, silencing C-CBL (P < 0.01) in the skeletal muscles of Creg1MKO mice significantly improved muscle regeneration induced by CTX injury (P < 0.01). CONCLUSIONS: Our findings suggest that CREG1 may be a potential therapeutic target for skeletal muscle regeneration.

The role of CREG1 in megakaryocyte maturation and thrombocytopoiesis.

Abnormal megakaryocyte maturation and platelet production lead to platelet-related diseases and impact the dynamic balance between hemostasis and bleeding. Cellular repressor of E1A-stimulated gene 1 (CREG1) is a glycoprotein that promotes tissue differentiation. However, its role in megakaryocytes remains unclear. In this study, we found that CREG1 protein is expressed in platelets and megakaryocytes and was decreased in the platelets of patients with thrombocytopenia. A cytosine arabinoside-induced thrombocytopenia mouse model was established, and the mRNA and protein expression levels of CREG1 were found to be reduced in megakaryocytes. We established megakaryocyte/platelet conditional knockout (Creg1pf4-cre) and transgenic mice (tg-Creg1). Compared to Creg1fl/fl mice, Creg1pf4-cre mice exhibited thrombocytopenia, which was mainly caused by inefficient bone marrow (BM) thrombocytopoiesis, but not by apoptosis of circulating platelets. Cultured Creg1pf4-cre-megakaryocytes exhibited impairment of the actin cytoskeleton, with less filamentous actin, significantly fewer proplatelets, and lower ploidy. CREG1 directly interacts with MEK1/2 and promotes MEK1/2 phosphorylation. Thus, our study uncovered the role of CREG1 in the regulation of megakaryocyte maturation and thrombopoiesis, and it provides a possible theoretical basis for the prevention and treatment of thrombocytopenia.

CREG1 stimulates AMPK phosphorylation and glucose uptake in skeletal muscle cells.

The cellular repressor of adenovirus early region 1A-stimulated gene 1 (CREG1) is a secreted glycoprotein involved in cell differentiation and energy metabolism. It also binds to insulin-like growth factor 2 receptor (IGF2R), a protein implicated in muscle regeneration. However, whether CREG1 regulates the regeneration and metabolism of skeletal muscles via IGF2R remains unclear. This study investigates the role of CREG1 in skeletal muscle regeneration and glucose uptake in C2C12 myotubes and a cardiotoxin (CTX)-induced mouse skeletal muscle regeneration model. CTX-treated skeletal muscle showed significantly higher levels of IGF2R, CREG1, phospho-AMPKα Thr172, and GLUT4 proteins. Similarly, treatment of myotubes with CREG1 also stimulated AMPKα phosphorylation and GLUT4 expression. CREG1-induced AMPKα phosphorylation and 2DG uptake in myotubes were suppressed by IGF2R knockdown and Compound C, an AMPK inhibitor. These results suggest that CREG1 stimulates glucose uptake in skeletal muscles partially through AMPK activation. Hence, CREG1 plays an essential role in muscle regeneration by affecting glucose metabolism in skeletal muscles.

Hepatocyte-specific deletion of cellular repressor of E1A-stimulated genes 1 exacerbates alcohol-induced liver injury by activating stress kinases.

Alcohol-associated liver disease (ALD) encompasses a wide range of pathologies from simple steatosis to cirrhosis and hepatocellular carcinoma and is a global health problem. Currently, there are no effective pharmacological treatments for ALD. We have previously demonstrated that aging exacerbates the pathogenesis of ALD, but the underlying mechanisms are still poorly understood. Cellular repressor of E1A-stimulated genes 1 protein (CREG1) is a recently identified small glycoprotein that has been implicated in aging process by promoting cellular senescence and activating stress kinases. Thus, the current study aimed to explore the role of aging associated CREG1 in ALD pathogenesis and CREG1 as a potential therapeutic target. Hepatic and serum CREG1 protein levels were elevated in ALD patients. Elevation of hepatic CREG1 protein and mRNA was also observed in a mouse model of Gao-binge alcohol feeding. Genetic deletion of the Creg1 gene in hepatocytes (Creg1∆hep ) markedly exacerbated ethanol-induced liver injury, apoptosis, steatosis and inflammation. Compared to wild-type mice, Creg1∆hep mice had increased phosphorylation of hepatic stress kinases such as apoptosis signal-regulating kinase 1 (ASK1), c-Jun N-terminal kinase (JNK) and p38 but not TGF-ß-activated kinase 1 (TAK1) or extracellular signal-regulated kinase (ERK) after alcohol feeding. In vitro, ethanol treatment elevated the phosphorylation of ASK1, JNK, and p38 in mouse hepatocyte AML-12 cells. This elevation was further enhanced by CREG1 knockdown but alleviated by CREG1 overexpression. Last, treatment with an ASK1 inhibitor abolished ethanol-induced liver injury and upregulated hepatic lipogenesis, proinflammatory genes and stress kinases in Creg1∆hep mice. Taken together, our data suggest that CREG1 protects against alcoholic liver injury and inflammation by inhibiting the ASK1-JNK/p38 stress kinase pathway and that CREG1 is a potential therapeutic target for ALD.

Flow cytometry-based study of model marine microalgal consortia revealed an ecological advantage of siderophore utilization by the dinoflagellate Amphidinium carterae.

Investigations of phytoplankton responses to iron stress in seawater are complicated by the fact that iron concentrations do not necessarily reflect bioavailability. Most studies to date have been based on single species or field samples and are problematic to interpret. Here, we report results from an experimental cocultivation model system that enabled us to evaluate interspecific competition as a function of iron content and form, and to study the effect of nutritional conditions on the proteomic profiles of individual species. Our study revealed that the dinoflagellate Amphidinium carterae was able to utilize iron from a hydroxamate siderophore, a strategy that could provide an ecological advantage in environments where siderophores present an important source of iron. Additionally, proteomic analysis allowed us to identify a potential candidate protein involved in iron acquisition from hydroxamate siderophores, a strategy that is largely unknown in eukaryotic phytoplankton.

CREG1 improves diet-induced obesity via uncoupling protein 1-dependent manner in mice.

Thermogenic brown and beige adipocytes express uncoupling protein 1 (UCP1) and stimulate energy metabolism, protecting against obesity and metabolic diseases such as type 2 diabetes and hyperlipidemia. Cellular repressor of E1A-stimulated genes 1 (CREG1) can stimulate thermogenic fat formation, induce UCP1, and reduce diet-induced obesity (DIO) in mice at normal room temperature. In this study, we investigated the effect of CREG1 administration and the importance of UCP1 in DIO inhibition under thermoneutral conditions at 30°C, which attenuate thermogenic fat formation. Interestingly, subcutaneous administration of recombinant CREG1 protein via an osmotic pump in C57BL/6J mice for four weeks increased UCP1 expression in interscapular brown adipose tissue (IBAT), inhibited visceral white fat hypertrophy with partial browning, and reduced DIO compared to that in PBS-treated mice. The mRNA expression of energy metabolism-related genes was significantly increased in the IBAT of CREG1-treated mice compared to that in PBS-treated mice. In contrast, adipocyte-specific overexpression of CREG1 failed to improve DIO in UCP1-knockout mice at thermoneutrality. Our results indicate the therapeutic potential of CREG1 administration for obesity under thermogenic fat-attenuating conditions and highlight the indispensable role of UCP1 in the DIO-inhibitory effect of CREG1.

CREG1 administration stimulates BAT thermogenesis and improves diet-induced obesity in mice.

Brown and beige adipocytes, which express thermogenic uncoupling protein-1 (UCP1), stimulate glucose and lipid metabolism, improving obesity and metabolic diseases such as type 2 diabetes and hyperlipidemia. Overexpression of cellular repressor of E1A-stimulated genes 1 (CREG1) promotes adipose tissue browning and inhibits diet-induced obesity (DIO) in mice. In this study, we investigated the effects of CREG1 administration on DIO inhibition and adipose browning. Subcutaneous administration of recombinant CREG1 protein to C57BL/6 mice stimulated UCP1 expression in interscapular brown adipose tissue (IBAT) and improved DIO, glucose tolerance and fatty liver compared with those in phosphate-buffered saline-treated mice. Injection of Creg1-expressing adenovirus into inguinal white adipose tissue (IWAT) significantly increased browning and mRNA expression of beige adipocyte marker genes compared with that in mice injected with control virus. The effect of Creg1 induction on beige adipocyte differentiation was supported in primary culture using preadipocytes isolated from IWAT of Creg1-transgenic mice compared with that of wild-type mice. Our results indicate a therapeutic effect of CREG1 on obesity and its associated pathology and a potential of CREG1 to stimulate brown/beige adipocyte formation.

CREG1 improves the capacity of the skeletal muscle response to exercise endurance via modulation of mitophagy.

CREG1 (cellular repressor of E1A-stimulated genes 1) is involved in tissue homeostasis and influences macroautophagy/autophagy to protect cardiovascular function. However, the physiological and pathological role of CREG1 in the skeletal muscle is not clear. Here, we established a skeletal muscle-specific creg1 knockout mouse model (creg1;Ckm-Cre) by crossing the Creg1-floxed mice (Creg1fl/fl) with a transgenic line expressing Cre recombinase under the muscle-specific Ckm (creatine kinase, muscle) promoter. In creg1;Ckm-Cre mice, the exercise time to exhaustion and running distance were significantly reduced compared to Creg1fl/fl mice at the age of 9 months. In addition, the administration of recombinant (re)CREG1 protein improved the motor function of 9-month-old creg1;Ckm-Cre mice. Moreover, electron microscopy images of 9-month-old creg1;Ckm-Cre mice showed that the mitochondrial quality and quantity were abnormal and associated with increased levels of PINK1 (PTEN induced putative kinase 1) and PRKN/PARKIN (parkin RBR E3 ubiquitin protein ligase) but reduced levels of the mitochondrial proteins PTGS2/COX2, COX4I1/COX4, and TOMM20. These results suggested that CREG1 deficiency accelerated the induction of mitophagy in the skeletal muscle. Mechanistically, gain-and loss-of-function mutations of Creg1 altered mitochondrial morphology and function, impairing mitophagy in C2C12 cells. Furthermore, HSPD1/HSP60 (heat shock protein 1) (401-573 aa) interacted with CREG1 (130-220 aa) to antagonize the degradation of CREG1 and was involved in the regulation of mitophagy. This was the first time to demonstrate that CREG1 localized to the mitochondria and played an important role in mitophagy modulation that determined skeletal muscle wasting during the growth process or disease conditions.Abbreviations: CCCP: carbonyl cyanide m-chlorophenylhydrazone; CKM: creatine kinase, muscle; COX4I1/COX4: cytochrome c oxidase subunit 4I1; CREG1: cellular repressor of E1A-stimulated genes 1; DMEM: dulbecco's modified eagle medium; DNM1L/DRP1: dynamin 1-like; FCCP: carbonyl cyanide p-trifluoro-methoxy phenyl-hydrazone; HSPD1/HSP60: heat shock protein 1 (chaperonin); IP: immunoprecipitation; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MFF: mitochondrial fission factor; MFN2: mitofusin 2; MYH1/MHC-I: myosin, heavy polypeptide 1, skeletal muscle, adult; OCR: oxygen consumption rate; OPA1: OPA1, mitochondrial dynamin like GTPase; PINK1: PTEN induced putative kinase 1; PPARGC1A/PGC-1α: peroxisome proliferative activated receptor, gamma, coactivator 1 alpha; PRKN/PARKIN: parkin RBR E3 ubiquitin protein ligase; PTGS2/COX2: prostaglandin-endoperoxide synthase 2; RFP: red fluorescent protein; RT-qPCR: real-time quantitative PCR; SQSTM1/p62: sequestosome 1; TFAM: transcription factor A, mitochondrial; TOMM20: translocase of outer mitochondrial membrane 20; VDAC: voltage-dependent anion channel.

Effects of CREG1 on Age-Associated Metabolic Phenotypes and Renal Senescence in Mice.

Cellular repressor of E1A-stimulated genes 1 (CREG1) is a secreted glycoprotein that accelerates p16-dependent cellular senescence in vitro. We recently reported the ability of CREG1 to stimulate brown adipogenesis using adipocyte P2-CREG1-transgenic (Tg) mice; however, little is known about the effect of CREG1 on aging-associated phenotypes. In this study, we investigated the effects of CREG1 on age-related obesity and renal dysfunction in Tg mice. Increased brown fat formation was detected in aged Tg mice, in which age-associated metabolic phenotypes such as body weight gain and increases in blood glucose were improved compared with those in wild-type (WT) mice. Blood CREG1 levels increased significantly in WT mice with age, whereas the age-related increase was suppressed, and its levels were reduced, in the livers and kidneys of Tg mice relative to those in WT mice at 25 months. Intriguingly, the mRNA levels of Ink4a, Arf, and senescence-associated secretory phenotype (SASP)-related genes and p38MAPK activity were significantly lowered in the aged kidneys of Tg mice, in which the morphological abnormalities of glomeruli as well as filtering function seen in WT kidneys were alleviated. These results suggest the involvement of CREG1 in kidney aging and its potential as a target for improving age-related renal dysfunction.