Repressor Of E1A-stimulated Genes Research Articles

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.

The secreted inhibitor of invasive cell growth CREG1 is negatively regulated by cathepsin proteases

Previous clinical and experimental evidence strongly supports a breast cancer-promoting function of the lysosomal protease cathepsin B. However, the cathepsin B-dependent molecular pathways are not completely understood. Here, we studied the cathepsin-mediated secretome changes in the context of the MMTV-PyMT breast cancer mouse model. Employing the cell-conditioned media from tumor-macrophage co-cultures, as well as tumor interstitial fluid obtained by a novel strategy from PyMT mice with differential cathepsin B expression, we identified an important proteolytic and lysosomal signature, highlighting the importance of this organelle and these enzymes in the tumor micro-environment. The Cellular Repressor of E1A Stimulated Genes 1 (CREG1), a secreted endolysosomal glycoprotein, displayed reduced abundance upon over-expression of cathepsin B as well as increased abundance upon cathepsin B deletion or inhibition. Moreover, it was cleaved by cathepsin B in vitro. CREG1 reportedly could act as tumor suppressor. We show that treatment of PyMT tumor cells with recombinant CREG1 reduced proliferation, migration, and invasion; whereas, the opposite was observed with reduced CREG1 expression. This was further validated in vivo by orthotopic transplantation. Our study highlights CREG1 as a key player in tumor–stroma interaction and suggests that cathepsin B sustains malignant cell behavior by reducing the levels of the growth suppressor CREG1 in the tumor microenvironment.

GW28-e1130 Cellular Repressor of E1A-stimulated Genes Promotes Smooth Muscle Cells Differentiation from Embryonic Stem Cells

GW28-e1130 Cellular Repressor of E1A-stimulated Genes Promotes Smooth Muscle Cells Differentiation from Embryonic Stem Cells

GW28-e1133 Cellular repressor of E1A-stimulated genes improves heart function in a mouse model of MI

GW28-e1133 Cellular repressor of E1A-stimulated genes improves heart function in a mouse model of MI

GW28-e1126 Cellular repressor of E1A-stimulated genes is a critical determinant of hypertensive vascular remodeling in response to angiotensin II

GW28-e1126 Cellular repressor of E1A-stimulated genes is a critical determinant of hypertensive vascular remodeling in response to angiotensin II

Abstract 361: Creg1 Interacts With Sec8 to Promote Cell-cell Adhesion During Cardiomyogenesis

Cellular repressor of E1A-stimulated genes 1 (CREG1) is a 24 kD glycoprotein essential for early embryonic development. Our immunofluorescence studies revealed that CREG1 is highly expressed at myocyte junctions in both embryonic and adult hearts. To explore it role in cardiomyogenesis, we employed gain- and loss-of-function analyses demonstrating that CREG1 is required for the differentiation of mouse embryonic stem (ES) cell into cohesive myocardium-like structures. Chimeric cultures of wild-type and CREG1 knockout ES cells expressing cardiac-specific reporters showed that the cardiomyogenic effect of CREG1 is cell autonomous. Furthermore, we identified a novel interaction between CREG1 and Sec8 of the exocyst complex, which tethers vesicles to the plasma membrane. Mutations of the amino acid residues D141 and P142 to alanine in CREG1 abolished its binding to Sec8. To address the role of the CREG1-Sec8 interaction in cardiomyogenesis, we rescued CREG1 knockout ES cells with wild-type and Sec8-binding mutant CREG1 and showed that CREG1 binding to Sec8 promotes cardiomyocyte differentiation and cohesion. Mechanistically, CREG1, Sec8 and N-cadherin all localize at cell-cell adhesion sites. CREG1 overexpression enhances the assembly of adherens and gap junctions. By contrast, its knockout inhibits the Sec8-N-cadherin interaction and induces their degradation. Finally, shRNA-mediated knockdown of Sec8 leads to cardiomyogenic defects similar to CREG1 knockout. These results suggest that the CREG1 binding to Sec8 enhances the assembly of intercellular junctions and promotes cardiomyogenesis.

Abstract 17066: Cellular Repressor of E1A-Stimulated Genes Protects From Myocardial Ischemia/Reperfusion Injury by Regulating Myocardial Autophagy and Apoptosis

Background: Cellular repressor of E1A-stimulated genes (Creg) is a secreted glycoprotein that regulates tissue and cell homeostasis and has been shown to antagonize heart injury; however, little is known about the role of CREG in ischemia/reperfusion injury (IRI). In this study, we aimed to investigate the role of CREG in cardiac IRI and clarify the mechanism. Methods: Myocardial ischemia/reperfusion (MIR) was established by ligation of left descending coronary artery for 30 min in wild type C57 mice (Creg+/+) and heterozygous Creg mice (Creg+/–). Expression of CREG was determined by Western Blot. Infarction size was determined by TTC and Evan’s Blue staining 24 hours after MIR. Cardiac function was evaluated by echocardiography at day 28. Recombinant CREG protein (0.3mg/kg·d) was supplemented through micro-pump embedded subcutaneously in Creg+/+ mice. Cardiac function and infarction size were assessed as mentioned above. Apoptosis, lysosome formation and function were detected in both heart tissue and cardiomyocyte cell line H9C2. Results: Level of CREG protein in mouse hearts was downregulated after mice were subjected to MIR, especially in Creg+/– mice. Creg+/– mice had larger infarction sizes after 24 hours of MIR treatment and worse cardiac function at day 28 compared with Creg+/+ mice. However, cardiac function was significantly improved in Creg+/+ mice treated with recombinant CREG protein. In Creg+/– mice, the number of cardiomyocytes undergoing apoptosis was increased, and LC3A and p62 accumulated, suggesting that autophagy was dysfunctional. Upregulation of CREG decreased apoptosis and activated autophagy of cardiomyocytes. Furthermore, we demonstrated that CREG protects cardiomyocytes against apoptosis by activating autophagy both in vivo and in vitro. Conclusions: CREG overexpression protects cardiac function against IRI by preventing cardiomyocyte apoptosis. The protective effects of CREG are partly mediated by activation of lysosomal autophagy.

ASSA14-03-28 Cellular repressor of E1A-stimulated genes improves heart function in a mouse model of MI

Backgroud Cellular repressor of E1A-stimulated genes (CREG) is a mannose-6-phosphate-containing secreted glycoprotein of 220 amino acids. It has been proposed that CREG acts as a ligand that enhances differentiation and/or reduces cell proliferation. In humans, the potential therapeutic role of embryonic stem cells (ESCs) in ischaemic heart disease is subject to intense investigation. Particularly, the contribution of ESCs to angiogenesis and cardiomyogenesis in myocardial ischemia is not well established. In our studies, we induced myocardial infarct (MI) in mouse model, and monitored the effects of ESCs transplantation of overexpression of CREG on cardiac function. Methods pCXN2-Flag-wtCREG, pCXN2-Flag-mutCREG and pCXN2-Flag-EGFP plasmids were transfected into ESCs by lipofectamine 2000. Coronary artery ligation to induce MI model in seven- to nine-week-old mice was developed by a novel and rapid surgical method. wtCREG, mutCREG and EGFP ESCs or DMEM were then injected into the peri-ischaemic area. Four groups of mice were analysed for haemodynamic and pathologic parameters 1 and 2 months after MI and injection. Results The heart weight to body weight ratio was also significantly decreased at day 28 (5.9 ± 0.5 and 5.5 ± 0.4) in comparison with control hearts (7.0 ± 0.5, p Conclusions Therefore, the expression of CREG improves cardiac functions and inhibits birosis and apoptosis. These data suggest that a key mechanism of the protective effects of ASK1 in reducing ischaemic injury is via maintaining the classic proapoptotic factor Bax in an inactive state.

ASSA14-03-20 Cellular repressor of E1A-stimulated genes protects against angiotensin II-induced vascular remodelling via degradation of angiotensin II type 1 receptor

Background Cellular repressor of E1A-stimulated genes (CREG), a new cardiovascular homeostasis regulator, has been proposed to reduce the neointimal formation after vascular injury via the maintenance of the quiescent mature vascular smooth muscle cell (VSMC) phenotype. We hypothesised that CREG is a negative regulator of angiotensin (Ang) II-mediated vascular remodelling. Methods Ten-week-old male heterozygous CREG-deficient (CREG +/- ) mice and their littermate wild-type (WT) mice were used. An osmotic minipump was implanted subcutaneously to infuse a dose of Ang II (1.5 mg/kg · d) or saline (vehicle) for 14 days. Ang II-infused WT mice were then treated with placebo or recombinant human CREG (rhCREG; 300 μg/kg · d). Systolic blood pressure (SBP); extent of vascular remodelling; protein level of collagen type I/III, angiotensin-converting enzyme-2 (ACE2), angiotensin receptor type 1 (AT1R) and Rab7 were evaluated. Primary VSMC culture was performed to assess AT1R and collagen protein expression before and after CREG gene transfection and knockdown. Results CREG levels are high in vascular media under basal conditions but rapidly decrease in response to Ang II. Ang II infusion for 14 days resulted that levels of SBP, intima medial thickness and vascular remodelling of the aorta and mesenteric artery were significantly greater in CREG +/- mice compared with the WT controls. Vascular gene expression level of CREG was lower in Ang II-treated CREG +/- mice than in WT mice. However, daily treatment of Ang II-infused WT mice with rhCREG blunted the increase of SBP. Ang II-mediated vascular remodelling and expression of collagen type I/III were also suppressed by rhCREG. Moreover, rhCREG treatment inhibited Ang II-mediated up-regulation of AT1R expression and down-regulation of ACE2 expression. Ang II-induced vascular remodelling was inhibited by rhCREG in association with reduced plasma Ang II and increased plasma Ang 1–7 levels. In adult VSMCs, CREG over-expression increased the expression of Rab7 by a regulatory mechanism of ubiquitination. Importantly, Ang II-mediated collagen production and AT1R expression was inhibited by CREG over-expression and augmented by CREG knock-down in a rab7-dependent manner. Conclusions Elevated Ang II induced hypertension and vascular remodelling, which were exacerbated by CREG deficiency, whereas rhCREG attenuated Ang II-induced adverse vascular remodelling. Hence, CREG is an important negative regulator of Ang II-induced vascular disease and suppresses adverse vascular remodelling.

GW25-e1138 Cellular repressor of E1A-stimulated genes protects against angiotensin II-induced hypertension and vascular remodeling via p38MAPK-mediated regulation of the renin-angiotensin system

GW25-e1138 Cellular repressor of E1A-stimulated genes protects against angiotensin II-induced hypertension and vascular remodeling via p38MAPK-mediated regulation of the renin-angiotensin system

CREG1 promotes angiogenesis and neovascularization.

Angiogenesis has long been considered as an important strategy for ischemic injury. It has been reported that cellular repressor of E1A-stimulated genes (CREG1) promotes human umbilical vein endothelial cell (HUVEC) proliferation, migration, and protects endothelial cell (EC) from apoptosis. However, its potential effect on angiogenesis remains undefined. In the present study, we investigated the role and mechanisms of CREG1 in promoting angiogenesis. We found that adenovirus-transduced CREG1 expression in HUVECs increases EC tube formation in matrigel and promotes neovascularization in matrigel plugs grafted into wild type mice. In addition, adenoviral CREG1 expression enhances filopodia formation, which is accompanied by increased expression of integrin-linked kinase (ILK) and activation of its downstream effector Cdc42. Hindlimb perfusion was significantly reduced after femoral artery ligation in CREG1 heterozygous knockout mice. Finally, adenoviral CREG1 was injected intramuscularly in gastrochemius and partially restores ischemic hindlimb perfusion. Our results demonstrated that CREG1 increases EC filopodia formation and vascular assembly via ILK-Cdc42 activation and promotes neovascularization, which might be a therapeutic target for ischemic injury.

ASSA13-10-14 Cellular Repressor of E1A-Stimulated Genes Accelerates Endothelial Angiogenesis Via Integrin-Linked Kinase-PINCH-CDC42 Activation

Background Cellular repressor of E1A-stimulated genes (CREG) is an important endothelial-protective gene, which is reported to modulate the mobility of endothelial cells. Objective The study aimed to investigate the effects of CREG on endothelial angiogenesis. Methods Aortic expression of CREG protein was detected by western blot and immnuostaining in CREG heterozygous (CREG +/- ) mice and wild-type littermates. Perfusion recovery of hind limb was valued by laser Doppler in mice and wild-type littermates 14 days after the hind limb arterial ligation. The matrigel experiments in vivo and in vitro were performed after the human umbilical vein endothelial cells (HUVEC) were infected by adenovirus overexpressing CREG or adenovirus expressing GFP as control, and the filopodium formation was observed to evaluate the CREG function on neovascularization. Different integrin-linked kinase (ILK) subunit site mutant plasmids and small interfering RNA identified p-Cdc42 were transfected into HUVEC to explore the mechanism through which CREG-mediated endothelial angiogenesis. Results Aortic expression of CREG protein was detected to reduce remarkably in CREG +/- mice compared to that in wild-type littermates. Meanwhile, laser Doppler perfusion imaging showed that perfusion recovery was significantly impaired in CREG +/- mice than that in wild-type littermates after 14 days hind limb arterial ligation (78.23 ± 7.2% vs 12.15 ± 2.058% in calf; 58.23 ± 6.5% vs 32.15 ± 3. 514% in thigh; P in vitro , enhanced neovascularization and improved limb perfusion in vivo , accompanied by filopodium formation and changes in cell shape. Mechanismly, integrin-linked kinase (ILK), a key adhesion plaque protein, participated in CREG-mediated endothelial angiogenesis. Furthermore, studies using small interfering RNA identified p-Cdc42 to be a key downstream molecule of ILK involved in CREG-mediated endothelial cell filopodium formation. Transfection with binding-site-mutant plasmids of ILK and co-immunoprecipitation revealed that CREG activated the ILK-PINCH complex, which is involved in the regulation of p-Cdc42 activation. Conclusions CREG overexpression stimulates the endothelial filopodium formation and regulates angiogenesis via the ILK/PINCH/p-Cdc42 signalling pathway, which provides the basis for future studies in the field of angiogenesis.

CREG mediated adventitial fibroblast phenotype modulation: A possible therapeutic target for proliferative vascular disease

Proliferative vascular diseases, of which neointimal formation is a key pathological feature, cause significant morbidity and mortality worldwide. Adventitia is the outermost connective tissue that surrounds an artery. In recent years, accumulating data indicate that adventitial fibroblasts participate in the formation of neointimal lesions. Our previous studies have demonstrated that cellular repressor of E1A-stimulated genes (CREG) plays critical roles in reducing neointimal hyperplasia by promoting vascular smooth muscle cell (VSMC) differentiation and grow arrest and inhibiting migration. Hence, it is plausible that genetical modification with CREG gene in adventitial fibroblasts might inhibit angiotensin II-induced transdifferentiation to myofibroblasts, proliferation and migration, as well as adventitial thickening, finally decreasing neointimal formation. Possible mechanisms may include CREG direct attenuation of reactive oxygen species derived from reduced cathepsin-dependent activation of NADPH oxidase and indirect suppression of the downstream of NADPH oxidase including ERK1/2, JNK and p38 MAPK pathways in adventitial fibroblasts. Therefore, CREG may be a potential therapeutic target for proliferative vascular diseases.

945. The Cellular Repressor of E1A-Stimulated Genes Regulate Cells Proliferation Mediated by IGFII and p42/44 MAPK in NIH3T3 Cells In Vitro

945. The Cellular Repressor of E1A-Stimulated Genes Regulate Cells Proliferation Mediated by IGFII and p42/44 MAPK in NIH3T3 Cells In Vitro

Application of laser capture microdissection and differential display technique for screening of pathogenic genes involved in endometrial carcinoma

The objective is to eliminate the interference from other cell types; gene fragments involved in endometrial carcinoma (EC) are screened and cloned. Human normal endometrial glandular epithelia and EC cells were harvested with laser capture microdissection (LCM). The purification and concentration of minimal RNA were used to screen differential expressed gene fragments involved in EC by fluoro differential display polymerase chain reaction (FDD-PCR). The differential gene fragments were cloned, sequenced, and then identified by reverse Northern blot hybridization. Positive fragments were analyzed with basic local alignment search tool (BLAST). Cyclin-dependent kinase 7 (CDK7) expressions in EC and normal endometrial tissue were tested by immunohistochemical staining. Of 38 differential bands, 3 bands were of high expression in normal endometrium and 35 in EC. Six positive differential gene fragments were obtained and BLAST analysis for them suggested that L1.1 was homologous (99% identical) to the CDK7; L1.9 had a 99% homology with protein phosphatase 1 regulatory (inhibitor) subunit 12 A (PPP1R12A); L1.21 and L1.22 showed a 100% homology with cellular repressor of E1A-stimulated genes 1 (CREG); and L1.25 and L1.26 indicated more than 98% homology with solute carrier family 39 (zinc transporter), member 10 (SLC39A10). Immunohistochemistry revealed that CDK7 expression was higher in EC than in normal endometrium. We conclude that pathogenic genes involved in EC are obtained with LCM and FDD-PCR. It has been first found that CDK7, PPP1R12A, CREG, and SLC39A10 are correlative with EC from gene level. CDK7 is strongly associated with EC and can be used as potential molecular marker of EC for further studies.

Expression of cellular repressor of E1A-stimulated genes in vascular smooth muscle cells of different phenotypes

The phenotypic modulation of vascular smooth muscle cells (VSMC) plays a central role in the pathogenesis of arteriosclerosis. The purpose of this study was to investigate the expression of the cellular repressor of E1A-activated genes (CREG) at the transcriptional and protein level in human internal thoracic artery smooth muscle cells (HITASY), which express different patterns of differentiation markers after serum withdrawal. After cloning and recombining the CREG vector, the antiserum against the CREG protein was produced from the rabbits immunized by the purification CREG protein. The specificity of purified polyclonal antibody was detected by Western blot assay. The DNA synthesis of HITASY cultured in serum-free and serum-supplemented medium was measured by the [(3)H]-thymidine incorporation. Western blot analysis detected the expression of smooth muscle-specific markers (smooth muscle alpha-actin, calponin). The localization of CREG in cells was examined with immunohistochemistry staining and expression of CREG mRNA and protein were analyzed by RT-PCR and Western blot in HITASY after serum withdrawal. The high specificity of polyclonal antibody against CREG obtained from rabbits was confirmed by Western blot assay. In response to serum withdrawal, cultured HITASY cells exhibited phenotypic conversion from synthetic into contractile one as evidenced by the data of [(3)H]-thymidine incorporation and Western blot. The DNA synthesis of HITASY precipitously dropped to background levels after serum withdrawal and nearly restored after reintroduction of serum to culture medium 2 days later. Western blot revealed a reversible upregulation of smooth muscle alpha-actin and calponin in HITASY after serum deprivation. Moreover, serum withdrawal also induced a prominent increase of CREG mRNA and protein expression which reached a peak on 3 days and decreased gradually on 5 approximately 7 days after serum withdrawal. Immunohistochemistry stain indicated the CREG protein mainly localizes in a perinuclear pattern in HITASY cells. Those data provide evidence that the coordinated changes in CREG gene and protein expression as well as smooth muscle-specific markers may take place in connection with the process of phenotypic modulation of VSMC in vitro.