Cellular Repressor Of E1A-stimulated Genes Research Articles

CREG1 deficiency impaired myoblast differentiation and skeletal muscle regeneration.

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. 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 100μL of 10μM 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. 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). Our findings suggest that CREG1 may be a potential therapeutic target for skeletal muscle regeneration.

Abstract 12306: CREG1 Attenuates Doxorubicin-Induced Cardiotoxicity via Inhibiting the Ferroptosis of Cardiomyocytes

Introduction: Doxorubicin (DOX) is currently one of the most used anthracycline anticancer drugs. However, the cardiotoxicity of DOX limits its application in cancer patients. Cellular repressor of E1A-stimulated genes (CREG1) is an important cardioprotective factor. Hypothesis: We investigated the underlying effects and mechanism of of CREG1 in DOX-induced cardiotoxicity have not been reported. Methods: In vivo, a mouse DOX-induced cardiotoxicity model was established by DOX intraperitoneal injection, the mRNA and protein expression of CREG1 in the myocardium was examined using real-time PCR and western blot. CREG1 transgenic mice and cardiac-specific CREG1 knockout mice were used to establish DOX-induced cardiotoxicity model. HE staining, Masson staining, WGA staining and western blot were applied to examine fibrosis, myocardial hypertrophy and ferroptosis. In vitro, neonatal mouse cardiomyocytes (NMCMs) were stimulated with DOX, CREG1 overexpression adenovirus and small interfering RNA was used to examine the role of CREG1 on the ferroptosis of NMCMs. Results: In vivo, the mRNA and protein expression of CREG1 were significantly reduced in DOX-treated myocardium. CREG1 overexpression alleviated the myocardial damage induced by DOX, and CREG1 deficiency aggravated the DOX-induced cardiotoxicity. The abnormal increase in ferroptosis was shown in the DOX-treated heart tissues. CREG1 overexpression inhibited the ferroptosis, and CREG1 deficiency aggravated the ferroptosis induced by DOX. In vitro, the mRNA and protein of CREG1 was reduced in DOX-treated NMCMs. CREG1 overexpression reduced the ferroptosis of cardiomyocytes, CREG1 knockdown aggravated the ferroptosis of cardiomyocytes induced by DOX. Mechanically, CREG1 inhibited the mRNA and protein expression of pyruvate dehydrogenase kinase 4 (PDK4). The effect of CREG1 overexpression on ferroptosis of cardiomyocytes was reversed by PDK4 overexpression. Conclusion: CREG1 alleviated DOX-induced cardiotoxicity by inhibiting ferroptosis of cardiomyocytes. Our findings might help clarify new roles of CREG1 in the development of DOX-induced cardiotoxicity.

Abstract 14526: CREG1 Deletion Inhibits Autophagy and Causes Cardiomyocyte Damage Under Nutritional Stress

Introduction: The cellular repressor of E1A-stimulated genes 1 (CREG1) is a small, highly conserved glycoprotein ubiquitously expressed in mammalian tissues. It localizes in the endosomal-lysosomal compartment and promotes lysosome biogenesis and acidification. The goal of the current study was to elucidate the physiological function of CREG1 in the mouse heart. Hypothesis: We hypothesized that deletion of Creg1 would result in pathological cardiac remodeling following nutritional stress. Methods: We generated whole-body and cardiomyocyte-specific Creg1 KO mouse lines via mating Creg1 fl/fl mice with UBC-Cre-ERT2 and Myh6-Cre mice, respectively. Mice were fasted for 16-40 hours, and hearts were evaluated by H&amp;E staining, immunofluorescence, electron microscopy, and biochemical analysis. Knockout mice were compared to within-litter controls. Results: Under ad lib feeding, some whole-body knockout mice exhibited myofibril disarray and irregular nuclei in the heart. After prolonged fasting, both whole-body and cardiomyocyte-specific deletion of Creg1 resulted in significantly increased intracellular vacuoles, pale cytoplasm, and loss of nuclei in the papillary muscles and interventricular septa ( Figure 1 ). Immunofluorescence staining demonstrated significantly increased size and number of LC3 puncta (an autophagosome marker), and Western blot demonstrated accumulation of LC3II in the Creg1 KO hearts. In Creg1 KO cardiomyocytes, electron microscopy revealed condensed and irregular mitochondria, which lost their registry with myofibers. In addition, mitophagy was impaired and sarcomeric Z-discs were either damaged or obliterated. Conclusion: CREG1 protects the heart from nutritional stress-induced injury, likely through the promotion of autophagic flux.

The CREG1-FBXO27-LAMP2 axis alleviates diabetic cardiomyopathy by promoting autophagy in cardiomyocytes

Autophagy plays an important role in the development of diabetic cardiomyopathy. Cellular repressor of E1A-stimulated genes 1 (CREG1) is an important myocardial protective factor. The aim of this study was to investigate the effects and mechanisms of CREG1 in diabetic cardiomyopathy. Male C57BL/6 J mice, Creg1 transgenic mice and cardiac-specific knockout mice were used to establish a type 2 diabetes model. Small animal ultrasound, Masson’s staining and western blotting were used to evaluate cardiac function, myocardial fibrosis and autophagy. Neonatal mouse cardiomyocytes (NMCMs) were stimulated with palmitate, and the effects of CREG1 on NMCMs autophagy were examined. CREG1 deficiency exacerbated cardiac dysfunction, cardiac hypertrophy and fibrosis in mice with diabetic cardiomyopathy, which was accompanied by exacerbated autophagy dysfunction. CREG1 overexpression improved cardiac function and ameliorated cardiac hypertrophy and fibrosis in diabetic cardiomyopathy by improving autophagy. CREG1 protein expression was decreased in palmitate-induced NMCMs. CREG1 knockdown exacerbated cardiomyocyte hypertrophy and inhibited autophagy. CREG1 overexpression inhibited cardiomyocyte hypertrophy and improved autophagy. LAMP2 overexpression reversed the effect of CREG1 knockdown on palmitate-induced inhibition of cardiomyocyte autophagy. CREG1 inhibited LAMP2 protein degradation by inhibiting the protein expression of F-box protein 27 (FBXO27). Our findings indicate new roles of CREG1 in the development of diabetic cardiomyopathy.

CREG Protects Retinal Ganglion Cells loss and Retinal Function Impairment Against ischemia-reperfusion Injury in mice via Akt Signaling Pathway.

The irreversible death of retinal ganglion cells (RGCs) plays an important role in the pathogenesis of glaucoma. Cellular repressor of E1A-stimulated genes (CREG), a secreted glycoprotein involved in cellular proliferation and differentiation, has been shown to protect against myocardial and renal ischemia-reperfusion damage. However, the role of CREG in retinal ischemia-reperfusion injury (RIRI) remains unknown. In this study, we aimed to explore the effect of CREG on RGCs apoptosis after RIRI. We used male C57BL/6J mice to establish the RIRI model. Recombinant CREG was injected at 1day before RIRI. The expression and distribution of CREG were examined by immunofluorescence staining and western blotting. RGCs survival was assessed by immunofluorescence staining of flat-mounted retinas. Retinal apoptosis was measured by the staining of TdT-mediated dUTP nick-end labeling and cleaved caspase-3. Electroretinogram (ERG)analysis and optomotor response were conducted to evaluate retinal function and visual acuity. The expressions of Akt, phospho-Akt (p-Akt), Bax, and Bcl-2 were analyzed by western blotting to determine the signaling pathways of CREG. We found that CREG expression was decreased after RIRI, and intravitreal injection of CREG attenuated RGCs loss and retinal apoptosis. Besides, the amplitudes of a-wave, b-wave, and photopic negative response (PhNR) in ERG, as well as visual function, were significantly restored after treatment with CERG. Furthermore, intravitreal injection of CREG upregulated p-Akt and Bcl-2 expression and downregulated Bax expression. Our results demonstrated that CREG protected RGCs from RIRI and alleviated retinal apoptosis by activating Akt signaling. In addition, CREG also improved retinal function and visual acuity.

Abstract 10586: CREG1-FBXO27-LAMP2 Axis Promotes Autophagy Function of Cardiomyocytes and Alleviates Diabetic Cardiomyopathy

Introduction: Diabetic cardiomyopathy is a chronic complication of diabetes in the absence of coronary artery disease, arterial hypertension and valvular disease. Autophagy plays an important role in the development of diabetic cardiomyopathy. Cellular repressor of E1A-stimulated genes 1 (CREG1) is an important myocardial protective factor. The aim of this study was to investigate the effects and mechanisms of CREG1 in diabetic cardiomyopathy. Methods: Male C57BL/6J mice, CREG1 transgenic mice, and CREG1 cardiac-specific knockout mice were used to establish a type 2 diabetic model. Small animal ultrasound, Masson’s staining, and western blotting were used to evaluate cardiac function, myocardial fibrosis and autophagy. Neonatal mouse cardiomyocytes (NMCMs) were isolated and cultured. NMCMs were infected with CREG1- overexpressing adenovirus or small interfering RNA, followed by stimulation with palmitic acid (PA), real-time PCR, western blotting and immunofluorescence staining were used. Results: CREG1 protein expression was decreased in the myocardium of diabetic cardiomyopathy. CREG1 deficiency in the heart aggravated cardiac dysfunction, cardiac hypertrophy, and fibrosis in mice with diabetic cardiomyopathy, accompanied by aggravated autophagy dysfunction. CREG1 overexpression improved cardiac function and ameliorated cardiac hypertrophy and fibrosis in diabetic cardiomyopathy, by improving autophagy. CREG1 protein expression was decreased in PA-stimulated NMCMs. CREG1 knockdown aggravated cardiomyocyte hypertrophy and inhibited autophagy, which were reversed by resveratrol treatment. Conversely, CREG1 overexpression inhibited cardiomyocyte hypertrophy and improved autophagy, which were reversed by chloroquine or bafilomycin A1 treatment. Overexpression of LAMP2 reversed the effect of CREG1 knockdown on the PA-induced inhibition of cardiomyocyte autophagy. CREG1 inhibited the LAMP2 protein degradation by inhibiting the expression of F-box protein 27 (FBXO27). Conclusions: CREG1-FBXO27-LAMP2 axis alleviated diabetic cardiomyopathy by promoting cardiomyocyte autophagy. Our findings might help clarify new roles of CREG1 in the development of diabetic cardiomyopathy.

Abstract P1026: Creg1 Inhibits Cardiomyocyte Proliferation And Promotes Myocardial Differentiation And Compaction During Postnatal Cardiac Development

Introduction: Overexpression of cellular repressor of E1A-stimulated genes 1 (CREG1) promotes the formation of compact, contracting myocardium-like structures during cardiomyogenesis from embryonic stem cells. Cell biology studies suggest that CREG1 promotes differentiation and inhibits proliferation of developing cardiomyocytes. This study aims to understand the role of CREG1 during myocardial development in vivo . Methods: Cardiac tissues were harvested from wild-type (WT) and constitutive Creg1 knockout (KO) postnatal day 1.5 and 17 through timed mating of heterozygous Creg1 mice. Creg1 KO mice were compared with Creg1 WT littermates using immunoblotting, immunofluorescence, and methylene blue staining. Results: Postnatal day 1.5 Creg1 KO hearts showed lower levels of expression of slow skeletal muscle troponin I (TnI) compared with WT counterparts. No significant difference in expression was seen between WT and KO Creg1 hearts for myosin light chain 3 (MLC3), myosin heavy chain (MHC), and cardiac troponin I. Immunofluorescence staining revealed increased numbers of PCNA (Proliferating Cell Nuclear Antigen) positive nuclei in postnatal day 1.5 Creg1 KO hearts, suggesting enhanced cardiomyocyte proliferation after loss of CREG1. Methylene blue staining in postnatal day 17 hearts displayed decreased myocardial compaction in Creg1 KO mice. Conclusion: CREG1 may promote cardiomyocyte differentiation and inhibit proliferation during postnatal myocardial development. Ablation of CREG1 results in cardiac compaction defects. Our findings suggest an important role for CREG1 in cardiac development.

CREG ameliorates embryonic stem cell differentiation into smooth muscle cells by modulation of TGF-β expression

Vascular smooth muscle cell (SMCs) differentiation is critical for cardiovascular development, but the mechanisms remain largely unknown. The overall aim of this study was to investigate the functional impact and mechanism of cellular repressor of E1A-stimulated genes (CREG) in SMC differentiation. Two embryonic stem cell (ESC) models were generated (1) the overexpression of CREG (CREG-OE), by transfection with Pcreg-IRECS2-EGFP vector, and (2) the knockout of CREG, by transfection with CREG shRNA (CREG-KO). Interesting, SMC-marker levels (SM α-actin, SM22, Calponin, and SM-MHC) dramatically increased in CREG-OE ESCs into the SMC while significantly decreased in CREG-KO ESCs during differentiation. After 14 days, and calcium ion concentrations in angiotensin II-stimulated embryoid bodies were increased in CREG-OE ESCs but reduced in CREG-KO ESCs. Consistently, the contractile capacity of SMC from CREG-OE ESC was increased, while the contractile capacity of SMC CREG1 from CREG-KO ESCs was significantly reduced. Furthermore, we demonstrated that CREG promotes differentiation of ESCs to SMCs and maturation of their function through the transforming growth factor-β -smad2/3 pathway.

CREG mitigates neonatal HIE injury through survival promotion and apoptosis inhibition in hippocampal neurons via activating AKT signaling.

Neonatal hypoxic ischemic encephalopathy (Neonatal HIE) is a common but serious disease caused by perinatal asphyxia injury in newborns. Elevated neuronal apoptosis plays an important role in the injury process post hypoxia ischemia of the brain, which accurate mechanism is still worthy to be studied. Cellular repressor of E1A-stimulated genes (CREG) possesses the protective effect in ischemia-reperfusion in multiple organs, including livers and hearts. The main purpose of this work was to investigate whether CREG was involved in alleviating neonatal HIE and explore the possible mechanisms. We found that CREG expression was downregulated in the hippocampus of neonatal HIE rats as well as oxygen-glucose deprivation/reperfusion (OGD/R)-treated hippocampal neurons. Besides, CREG overexpression promoted survival while inhibited apoptosis in OGD/R-induced hippocampal neurons accompanied by AKT signaling activation, which could be reversed by CREG silence. In addition, the protective effects of CREG overexpression could be antagonized by AKT deactivation, indicating the function of CREG was attributed by regulating AKT pathway. Collectedly, we demonstrated that CREG protected hippocampal neurons from hypoxic ischemia-induced injury through regulating survival and apoptosis via activating AKT signaling pathway.

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-β-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.

A novel function of CREG in metabolic disorders.

Metabolic disorders are public health problems that require prevention and new efficient drugs for treatment. Cellular repressor of E1A-stimulated genes (CREG) is ubiquitously expressed in mature tissues and cells in mammals and plays a critical role in keeping cells or tissues in a mature, homeostatic state. Recently, CREG turns to be an important mediator in the development ofmetabolic disorders. Here in this review, we briefly discuss the structure and molecular regulation of CREG along with the therapeutic strategy to combat the metabolic disorders.

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

ABSTRACT 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.

CREG ameliorates the phenotypic switching of cardiac fibroblasts after myocardial infarction via modulation of CDC42

Phenotype switching of cardiac fibroblasts into myofibroblasts plays important role in cardiac fibrosis following myocardial infarction (MI). Cellular repressor of E1A-stimulated genes (CREG) protects against vascular and cardiac remodeling induced by angiotensin-II. However, the effects and mechanisms of CREG on phenotype switching of cardiac fibroblasts after MI are unknown. This study aimed to investigate the role of CREG on the phenotype switching of cardiac fibroblasts following MI and its mechanism. Our findings demonstrated that, compared with littermate control mice, cardiac function was deteriorated in CREG+/− mice on day 14 post-MI. Fibrosis size, αSMA, and collagen-1 expressions were increased in the border regions of CREG+/− mice on day 14 post-MI. Conversely, exogenous CREG protein significantly improved cardiac function, inhibited fibrosis, and reduced the expressions of αSMA and collagen-1 in the border regions of C57BL/6J mice on day 14. In vitro, CREG recombinant protein inhibited αSMA and collagen-1 expression and blocked the hypoxia-induced proliferation and migration of cardiac fibroblasts, which was mediated through the inhibition of cell division control protein 42 (CDC42) expression. Our findings could help in establishing new strategies based on the clarification of the role of the key molecule CREG in phenotype switching of cardiac fibroblasts following MI.

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.

CREG improves cardiac function by regulating cardiomyocytes' autophagy in diabetic myocardial infarction rats.

We aimed to observe the changes of cardiac function, cell morphology, cellular repressor of E1A-stimulated genes (CREG) and LC3-II after myocardial infarction (MI) in non-diabetic and diabetic rats, and to explore the relationship between myocardial damage and CREG and autophagy in diabetic rats. Diabetic rat models were prepared by intraperitoneal injection of low concentration (50 mg/kg) streptozotocin (STZ). MI models were established in normal rats and diabetic rats. The cardiac function of each group was detected by echocardiography. The pathological results of myocardial tissue in the infarcted area were observed under light microscope. The expression of CREG was detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Myocardial protein CREG LC3-II expression was measured by Western blot. Autophagy levels were also detected at the cellular level. Construction of CREG overexpressing adenovirus transfected H9c2 cells, and injection of rats with AAV-CREG to achieve the purpose of overexpressing CREG. In vitro and in vivo experiments were conducted to explore the effects of CREG on autophagy and cardiac function in a diabetic MI model. Compared with the non-diabetic sham (NS) group, there were no marked differences in cardiac function and CREG levels in the diabetic sham (DS) group. Compared with the NS group, the cardiac function of the non-diabetic myocardial infarction (NI) group and the diabetic infarction myocardial (DI) group were reduced, and the levels of autophagy were increased. However, the cardiac function of the DI group was worse than that of the NI group, and the autophagy level of the DI group was lower than the NI group. The results at the cellular level were similar to the experiments in vivo. The overexpression of CREG in vivo or in vitro can increase autophagy levels and improve cardiac function. The exacerbation of myocardial injury after MI in diabetic rats may be related to the inhibition of CREG in myocardial cells of infarcted area by diabetes.

Recombinant Cellular Repressor of E1A-Stimulated Genes Protects against Renal Fibrosis in Dahl Salt-Sensitive Rats

Background: Human cellular repressor of E1A-stimulated genes (CREG) is a secreted glycoprotein that attenuates angiotensin II-induced hypertension, alleviates myocardial fibrosis, and improves heart function. However, the role of CREG in high-salt (HS) diet-induced hypertensive nephropathy is unclear. Methods: To determine the effects and molecular mechanisms of CREG in HS diet-induced hypertensive nephropathy, we established a hypertensive nephropathy animal model in Dahl salt-sensitive (SS) rats fed a HS diet (8% NaCl, n = 20) for 8 weeks. At week 4 of HS loading, these rats were administered recombinant CREG (reCREG; 35 µg/kg·day, n = 5) and saline (n = 5) via subcutaneously implanted pumps and were also administered the vasodilator hydralazine (20 mg/kg·day, n = 5) in drinking water. We used hematoxylin and eosin staining, Masson’s trichrome staining, immunohistochemical labeling, western blotting, RT-PCR, and Tunel staining to determine the signaling pathways of CREG in HS diet-induced hypertensive nephropathy. Results: After 8 weeks of HS intake, the Dahl SS rats developed renal dysfunction and severe renal fibrosis associated with reductions of 78 and 67% in CREG expression, respectively, at both mRNA and protein levels in the kidney. Administration of reCREG improved renal function and relieved renal fibrosis. Administration of CREG also inhibited monocyte infiltration and reduced apoptosis in the kidney cells. CREG overexpression upregulated forkhead box P1 expression and inhibited the transforming growth factor-β1 signaling pathway. Conclusion: Our study shows that CREG protected the kidney against HS-diet-induced renal damage and provides new insights into the mechanisms underlying kidney injury.

Characterising the mechanisms underlying genetic resistance to amoebic gill disease in Atlantic salmon using RNA sequencing

BackgroundGill health is one of the main concerns for Atlantic salmon aquaculture, and Amoebic Gill Disease (AGD), attributable to infection by the amoeba Neoparamoeba perurans, is a frequent cause of morbidity. In the absence of preventive measures, increasing genetic resistance of salmon to AGD via selective breeding can reduce the incidence of the disease and mitigate gill damage. Understanding the mechanisms leading to AGD resistance and the underlying causative genomic features can aid in this effort, while also providing critical information for the development of other control strategies. AGD resistance is considered to be moderately heritable, and several putative QTL have been identified. The aim of the current study was to improve understanding of the mechanisms underlying AGD resistance, and to identify putative causative genomic factors underlying the QTL. To achieve this, RNA was extracted from the gill and head kidney of AGD resistant and susceptible animals following a challenge with N. perurans, and sequenced.ResultsComparison between resistant and susceptible animals primarily highlighted differences mainly in the local immune response in the gill, involving red blood cell genes and genes related to immune function and cell adhesion. Differentially expressed immune genes pointed to a contrast in Th2 and Th17 responses, which is consistent with the increased heritability observed after successive challenges with the amoeba. Five QTL-region candidate genes showed differential expression, including a gene connected to interferon responses (GVINP1), a gene involved in systemic inflammation (MAP4K4), and a positive regulator of apoptosis (TRIM39). Analyses of allele-specific expression highlighted a gene in the QTL region on chromosome 17, cellular repressor of E1A-stimulated genes 1 (CREG1), showing allelic differential expression suggestive of a cis-acting regulatory variant.ConclusionsIn summary, this study provides new insights into the mechanisms of resistance to AGD in Atlantic salmon, and highlights candidate genes for further functional studies that can further elucidate the genomic mechanisms leading to resistance and contribute to enhancing salmon health via improved genomic selection.

MicroRNA miR-505-5p Promotes Oxygen-Glucose Deprivation/Reoxygenation-Induced Neuronal Injury via Negative Regulation of CREG1 in Cultured Neuron-Like Cells

Cerebral ischemia/reperfusion (I/R) injury is associated with various cardiovascular and cerebrovascular diseases with high disability, morbidity, and mortality rates. MicroRNAs (miRNAs) are related to the pathogenesis of the above diseases. MiR-505-5p, a kind of such miRNAs, was found to be highly expressed in cerebral I/R injuries, but the mechanism of action of this miRNA in cerebral I/R injury was unclear. In our study, up-regulation of miR-505-5p was detected in cultured oxygen-glucose deprivation/reoxygenation (OGD/R)-subjected PC12 and HEC293 cells. Silencing of miR-505-5p in OGD/R-affected neuron-like cells not only decreased the OGD/R-injury (according to cell viability, SOD increase, and LDH and MDA decreases), but also reduced apoptosis (decreasing the cleaved caspase-3 and PARP protein levels). Interestingly, expression of CREG1 (Cellular Repressor of E1Astimulated Genes 1) was low in OGD/R-subjected neurons; it was verified as a possible target gene of miR-505-5p. CREG1 knockdown can reverse the effect of miR-505-5p silencing in OGD/R-induced neural injury. Taken together, the data obtained provide a new target for cerebral I/R injury treatment.

Epoxyeicosatrienoic intervention improves NAFLD in leptin receptor deficient mice by an increase in HO-1-PGC1α mitochondrial signaling

BackgroundNon-alcoholic fatty liver disease (NAFLD) is associated with obesity and is considered to be an inflammatory disorder characterized by fatty acid accumulation, oxidative stress, and lipotoxicity. We have previously reported that epoxyeicosatrienoic acid-agonist (EET-A) has multiple beneficial effects on cardiac, renal and adipose tissue function while exhibiting both anti-inflammatory and anti-oxidant activities. We hypothesized that EET-A intervention would play a central role in attenuation of obesity-induced steatosis and hepatic fibrosis that leads to NAFLD. MethodsWe studied the effect of EET-A on fatty liver using db/db mice as a model of obesity. Mice were fed a high fat diet (HFD) for 16 weeks and administered EET-A twice weekly for the final 8 weeks. Resultsdb/db mice fed HFD significantly increased hepatic lipid accumulation as manifested by increases in NAS scores, hepatic fibrosis, insulin resistance, and inflammation, and decreases in mitochondrial mitofusin proteins (Mfn 1/2) and anti-obesity genes Fibroblast growth factor 21 (FGF21) and Cellular Repressor of E1A-Stimulated Genes 1 (CREG1). EET-A administration reversed the decrease in these genes and reduced liver fibrosis. Knockout of Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) in EET-A treated mice resulted in a reversal of the beneficial effects of EET-A administration. ConclusionsEET-A intervention diminishes fatty acid accumulation, fibrosis, and NFALD associated with an increase in HO-1-PGC1α and increased insulin receptor phosphorylation. A pharmacological strategy involving EETs may offer a potential therapeutic approach in preventing fibrosis, mitochondrial dysfunction, and the development of NAFLD.