Iguratimod suppresses sclerostin and receptor activator of NF-κB ligand production via the extracellular signal-regulated kinase/early growth response protein 1/tumor necrosis factor alpha pathway in osteocytes and ameliorates disuse osteoporosis in mice.

Disuse osteoporosis is a prevalent complication among patients afflicted with rheumatoid arthritis (RA). Although reports have shown that the antirheumatic drug iguratimod (IGU) ameliorates osteoporosis in RA patients, details regarding its effects on osteocytes remain unclear. The current study examined the effects of IGU on osteocytes using a mouse model of disuse-induced osteoporosis, the pathology of which crucially involves osteocytes. A reduction in distal femur bone mass was achieved after 3 weeks of hindlimb unloading in mice, which was subsequently reversed by intraperitoneal IGU treatment (30 mg/kg; five times per week). Histology revealed that hindlimb-unloaded (HLU) mice had significantly increased osteoclast number and sclerostin-positive osteocyte rates, which were suppressed by IGU treatment. Moreover, HLU mice exhibited a significant decrease in osteocalcin-positive cells, which was attenuated by IGU treatment. In vitro, IGU suppressed the gene expression of receptor activator of NF-κB ligand (RANKL) and sclerostin in MLO-Y4 and Saos-2 cells, which inhibited osteoclast differentiation of mouse bone marrow cells in cocultures. Although IGU did not affect the nuclear translocation or transcriptional activity of NF-κB, RNA sequencing revealed that IGU downregulated the expression of early growth response protein 1 (EGR1) in osteocytes. HLU mice showed significantly increased EGR1- and tumor necrosis factor alpha (TNFα)-positive osteocyte rates, which were decreased by IGU treatment. EGR1 overexpression enhanced the gene expression of TNFα, RANKL, and sclerostin in osteocytes, which was suppressed by IGU. Contrarily, small interfering RNA-mediated suppression of EGR1 downregulated RANKL and sclerostin gene expression. These findings indicate that IGU inhibits the expression of EGR1, which may downregulate TNFα and consequently RANKL and sclerostin in osteocytes. These mechanisms suggest that IGU could potentially be used as a treatment option for disuse osteoporosis by targeting osteocytes.

Investigating the synergy of Shikonin and Valproic acid in inducing apoptosis of osteosarcoma cells via ROS-mediated EGR1 expression.

BACKGROUND: Osteosarcoma is the most prevalent malignant bone tumour with a poor prognosis. Shikonin (SHK) is derived from the traditional Chinese medicine Lithospermum that has been extensively studied for its notable anti-tumour effects, including for osteosarcoma. However, its application has certain limitations. Valproic acid (VPA) is a histone deacetylase inhibitor (HDACI) that has recently been employed as an adjunctive therapeutic agent that allows chromatin to assume a more relaxed state, thereby enhancing anti-tumour efficacy. PURPOSE: This study was aimed to investigate the synergistic anti-tumour efficacy of SHK in combination with VPA and elucidate its underlying mechanism. METHODS/STUDY DESIGN: CCK-8 assays were utilized to calculate the combination index. Additional assays, including colony formation, acridine orange/ethidium bromide double fluorescent staining, and flow cytometry, were employed to evaluate the effects on osteosarcoma cells. Wound healing and transwell assays were utilized to assess cell mobility. RNA sequencing, PCR, and Western blot analyses were conducted to uncover the underlying mechanism. Rescue experiments were performed to validate the mechanism of apoptotic induction. The impact of SHK and VPA combination treatment on primary osteosarcoma cells was also assessed. Finally, in vivo experiments were conducted to validate its anti-tumour effects and mechanism. RESULTS: The combination of SHK and VPA synergistically inhibited the proliferation and migration of osteosarcoma cells in vitro and induced apoptosis in these cells. Through a comprehensive analysis involving RNA sequencing, PCR, Western blot, and rescue experiments, we have substantiated our hypothesis that the combination of SHK and VPA induced apoptosis via the ROS-EGR1-Bax axis. Importantly, our in vivo experiments corroborated these findings, demonstrating the potential of the SHK and VPA combination as a promising therapeutic approach for osteosarcoma. CONCLUSION: The combination of SHK and VPA exerted an anti-tumour effect by inducing apoptosis through the ROS-EGR1-Bax pathway. Repurposing the old drug VPA demonstrated its effectiveness as an adjunctive therapeutic agent for SHK, enhancing its anti-tumour efficacy and revealing its potential value. Furthermore, our study expanded the application of natural compounds in the anti-tumour field and overcame some of their limitations through combination therapy. Finally, we enhanced the understanding of the mechanistic pathways linking reactive oxygen species (ROS) accumulation and apoptosis in osteosarcoma cells. Additionally, we elucidated the role of EGR1 in osteosarcoma cells, offering novel strategies and concepts for the treatment of osteosarcoma.

EGR1 induces EMT in pancreatic cancer via a P300/SNAI2 pathway.

BACKGROUND: The prognosis of pancreatic cancer patients remains relatively poor. Although some patients would receive surgical resection, distant metastasis frequently occurs within one year. Epithelial-mesenchymal transition (EMT), as a pathological mechanism in cancer progression, contributed to the local and distant metastasis of pancreatic cancer. METHODS: Tissue microarray analysis and immunohistochemistry assays were used to compare the expression of EGR1 in pancreatic cancer and normal pancreatic tissues. Transwell chambers were used to evaluated the migration and invasion ability of cancer cells. Immunofluorescence was utilized to assess the expression of E-cadherin. ChIP-qPCR assay was applied to verify the combination of EGR1 and SNAI2 promoter sequences. Dual-luciferase reporter assay was used to detect the gene promoter activation. Co-IP assay was conducted to verify the interaction of EGR1 and p300/CBP. RESULTS: EGR1 was highly expressed in pancreatic cancer rather than normal pancreatic tissues and correlated with poor prognosis and cancer metastasis. EGR1 was proved to enhance the migration and invasion ability of pancreatic cells. Besides, EGR1 was positively correlated with EMT process in pancreatic cancer, via a SNAI2-dependent pathway. P300/CBP was found to play an auxiliary role in the transcriptional activation of the SNAI2 gene by EGR1. Finally, in vivo experiments also proved that EGR1 promoted liver metastasis of pancreatic cancer. CONCLUSION: Our findings implied the EMT-promoting effect of EGR1 in pancreatic cancer and revealed the intrinsic mechanism. Blocking the expression of EGR1 may be a new anticancer strategy for pancreatic cancer.

The involvement of EGR1 in neuron apoptosis in the in vitro model of spinal cord injury via BTG2 up-regulation.

OBJECTIVE: EGR1 has been implicated in the progression of spinal cord injury (SCI). Nevertheless, its specific mechanism in SCI remains to be investigated. Hence, this study explored the potential mechanism of EGR1 in SCI by focusing on neuron apoptosis. METHODS: H2O2 was utilized to treat rat neurons-dorsal spinal cord (RN-dsc) for the construction of an in vitro model of SCI. Afterwards, cell survival, apoptosis, and LDH leakage were detected to evaluate the injury degree of H2O2-treated RN-dsc. The expression of apoptosis-related proteins was also measured. Additionally, EGR1 was silenced and/or BTG2 was overexpressed in RN-dsc before H2O2 treatment to assess the impacts of EGR1 and BTG2 on H2O2-induced RN-dsc. Jasper online website was utilized to predict binding sites of EGR1 on BTG2, and dual-luciferase reporter gene and chromatin immunoprecipitation (ChIP) assays were utilized to verify the binding between EGR1 and BTG2. RESULTS: H2O2 treatment suppressed survival and promoted apoptosis in RN-dsc, accompanied by upregulated LDH, Bax, and cleaved-caspase-3 and down-regulated Bcl-2. Moreover, EGR1 and BTG2 were up-regulated in H2O2-induced RN-dsc. Mechanistically, EGR1 was bound to the promoter of BTG2 to transcriptionally activate BTG2. EGR1 knockdown diminished apoptosis and LDH, Bax, and cleaved-caspase-3 levels while elevating survival and Bcl-2 levels in H2O2-induced RN-dsc. These effects of EGR1 knockdown were abrogated by further BTG2 overexpression. DISCUSSION: Conclusively, EGR1 promotes H2O2-induced apoptosis in RN-dsc by activating BTG2 transcription.

Hepatocyte Ninjurin2 promotes hepatic stellate cell activation and liver fibrosis through the IGF1R/EGR1/PDGF-BB signaling pathway.

BACKGROUND: Liver fibrogenesis is orchestrated by the paracrine signaling interaction between several resident cell types regulating the activation of hepatic stellate cells (HSCs). However, the molecular mechanisms underlying paracrine regulation are largely unknown. The aim of this study is to elucidate the role of Ninjurin2 in the crosstalk between hepatocytes and HSCs and better understand the implications of Ninjurin2 in liver fibrosis. METHODS: Ninj2 knockout mice (Ninj2-/-) and hepatocyte-specific Ninj2 overexpression mice (Ninj2Hep-tg) were constructed and followed by the induction of liver fibrosis using methionine- and choline-deficient (MCD) diet. The relationship between Ninjurin2 and liver fibrosis phenotype was evaluated in vivo by measurement of fibrotic markers and related genes. We used an in vitro transwell cell co-culture model to examine the impact of Ninjurin2 in hepatocytes on the crosstalk to HSCs. The interaction of Ninjurin2 and IGF1R and the regulation of PI3K-AKT-EGR1 were analyzed in vivo and in vitro. Finally, an inhibitory Ninjurin2 peptide was injected intravenously via the tail vein to investigate whether inhibiting of Ninjurin2 cascade can attenuate MCD diet-induced liver fibrosis in mice. RESULTS: We found that hepatic Ninjurin2 expression was significantly increased in fibrotic human liver and MCD diet-induced liver injury mouse models. In the mouse model, hepatocyte-specific overexpression of Ninj2 exacerbates MCD-induced liver fibrosis, while global Ninj2 knockout reverses the phenotype. To mimic hepatocyte-HSC crosstalk during liver fibrosis, we used co-culture systems containing hepatocytes and HSCs and determined that Ninjurin2 overexpression in hepatocytes directly activates HSCs in vitro. Mechanistically, Ninjurin2 directly interacts with insulin-like growth factor 1 receptor (IGF1R) and increases the hepatocyte secretion of the fibrogenic cytokine, platelet-derived growth factor-BB (PDGF-BB) through IGF1R-PI3K-AKT-EGR1 cascade. Inhibition of PDGFRB signaling in HSCs can abolish the profibrogenic effect of Ninjurin2. In addition, we demonstrated that a specific inhibitory Ninjurin2 peptide containing an N-terminal adhesion motif mitigates liver fibrosis and improves hepatic function in the mouse models by negatively regulating the sensitivity of IGF1R to IGF1 in hepatocytes. CONCLUSION: Hepatic Ninjurin2 plays a key role in liver fibrosis through paracrine regulation of PDGF-BB/PDGFRB signaling in HSCs, and the results suggesting Ninjurin2 may be a potential therapeutic target.

LncRNA 1500026H17Rik knockdown ameliorates high glucose-induced mouse podocyte injuries through the miR-205-5p/EGR1 pathway.

BACKGROUND: Podocyte injuries and dysfunctions contribute to the development of diabetic nephropathy (DN). This study aimed to investigate the role and novel mechanism of lncRNA 1500026H17Rik in high glucose (HG)-treated podocytes. METHODS: DN mice were induced by streptozotocin, and DN in vitro models were constructed in mouse podocytes treated with HG. The expression of fibrosis-related proteins and early growth response protein 1 (EGR1) was detected by western blot. The expression of 1500026H17Rik, miR-205-5p and EGR1 mRNA was measured by quantitative real-time polymerase chain reaction (qRT-PCR). Cell apoptosis was monitored by flow cytometry assay. Oxidative stress was assessed according to the levels of superoxide dismutase (SOD), malondialdehyde (MDA) and reactive oxygen species (ROS). Inflammatory response was assessed according to the releases of interleukin-6 (IL-6), interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α). The target relationship between miR-205-5p and 1500026H17Rik or EGR1 was validated by dual-luciferase reporter assay, RNA immunoprecipitation (RIP) assay and RNA pull-down assay. RESULTS: 1500026H17Rik was upregulated in DN mice and HG-induced podocytes. 1500026H17Rik knockdown alleviated podocyte apoptosis, fibrosis, oxidative stress and inflammation induced by HG. MiR-205-5p was a target of 1500026H17Rik, and EGR1 was a downstream target of miR-205-5p. Rescue experiments presented that miR-205-5p inhibition reversed the effects of 1500026H17Rik knockdown. Moreover, miR-205-5p restoration also ameliorated HG-induced cell injuries, which were overturned by EGR1 overexpression. In addition, EGR1 overexpression recovered podocyte apoptosis, fibrosis, oxidative stress and inflammation weakened by 1500026H17Rik knockdown. CONCLUSION: 1500026H17Rik knockdown alleviated HG-induced podocyte injuries, including apoptosis, fibrosis, oxidative stress and inflammation, by governing the miR-205-5p/EGR1 pathway, thus involving in DN development.

Acidosis-Induced Regulation of Egr1 and Ccn1 In Vitro and in Experimental Tumours In Vivo.

Extracellular acidosis is a characteristic of solid tumours, resulting from hypoxia-induced glycolytic metabolism as well as from the "Warburg effect" (aerobic glycolysis). The acidic environment has shown to affect functional tumour properties (proliferation, migration, invasion) and thus the aim of the study was to identify signalling mechanisms, mediating these pH-dependent effects. Therefore, the serum response factor (Srf) and the activation of the serum response element (SRE) by acidosis were analysed in AT-1 prostate carcinoma cells. Furthermore, the expression of downstream targets of this cascade, namely the early growth response 1 (Egr1), which seems to be involved in tumour proliferation, and the cellular communication network factor 1 (Ccn1), which both contain SRE in their promotor region were examined in two tumour cell lines. Extracellular acidification led to an upregulation of Srf and a functional activation of the SRE. Egr1 expression was increased by acidosis in AT-1 cells whereas hypoxia had a suppressive effect. In experimental tumours, in vivo Egr1 and Ccn1 were also found to be acidosis-dependent. Also, it turned out that pH regulated expression of Egr1 was followed by comparable changes of p21, which is an important regulator of the cell cycle.This study identifies the Srf-SRE signalling cascade and downstream Egr1 and Ccn1 to be acidosis-regulated in vitro and in vivo, potentially affecting tumour progression. Especially linked expression changes of Egr1 and p21 may mediate acidosis-induced effects on cell proliferation.

FRZB is Regulated by the Transcription Factor EGR1 and Inhibits the Growth and Invasion of Triple-Negative Breast Cancer Cells by Regulating the JAK/STAT3 Pathway.

BACKGROUND: To explore the expression of frizzled related protein (FRZB) in triple-negative breast cancer (TNBC) and role of FRZB in TNBC cell growth and invasion. METHODS: Breast cancer clinical data were downloaded from the Cancer Genome Atlas. FRZB and early growth response 1 (EGR1) mRNA levels in TNBC were measured by quantitative real-time polymerase chain reaction. FRZB protein level was measured by immunohistochemistry and western blot. Proliferation, migration, and invasion of TNBC cells were detected by colony formation, wound healing, and transwell assay, respectively. The protein levels of EGR1, E-cadherin, N-cadherin, Snail, p-JAK1/JAK1, p-JAK2/JAK2, and p-STAT3/STAT3 were measured by western blot. JASPAR was used to predict the binding site of FRZB and EGR1. The binding ability of FRZB and EGR1 was verified by dual-luciferase reporter gene assay and chromatin immunoprecipitation assay. RESULTS: FRZB was low expressed in TNBC tissues and cells. Silencing FRZB promoted cell proliferation, migration, invasion, and EMT and activated JAK/STAT pathway in MDA-MB-468 and MDA-MB-231 cells, but overexpression of FRZB acted opposite effects in MDA-MB-468 and MDA-MB-231 cells. EGR1 was low expressed in TNBC samples and positively correlated with FRZB. Moreover, EGR1 could recover the promotion of silencing FRZB on cell proliferation, migration, invasion, and JAK/STAT pathway in MDA-MB-468 cells, but silencing EGR1 led to the opposite results in MDA-MB-231 cells. CONCLUSION: FRZB was low expressed in TNBC and was regulated by EGR1, and FRZB inhibited TNBC cell growth and invasion by regulating the JAK/STAT3 pathway.

Egr1 confers protection against acetaminophen­induced hepatotoxicity via transcriptional upregulating of Acaa2.

Background : Acetaminophen (APAP)-induced liver injury (AILI) is a common cause of drug-induced liver injury (DILI). The mechanism underlying protection in AILI or DILI remains to be elucidated, and the role of early growth response 1 (Egr1) in AILI and potential mechanisms remain to be known. Methods : The role of Egr1 was studied both in vivo and in vitro. Liver-specific Egr1-knockout (Egr1LKO) mice and those overexpressing Egr1 via tail vein injection of Egr1-expressing adenovirus (Ad-Egr1) were utilized with AILI. Chromatin immunoprecipitation-sequencing, RNA-sequencing, seahorse XF analysis, and targeted fatty acid analysis were performed. EGR1 levels were also studied in liver tissues and serum samples from AILI/DILI patients. Results: In this study, we have demonstrated that Egr1 was upregulated in AILI models in vivo and in vitro. liver-specific Egr1 knockout aggravated AILI; however, Ad-Egr1 treatment ameliorated this. Mechanistically, Egr1 deficiency inhibited, whereas overexpression promoted, mitochondrial respiratory function and fatty acid ß-oxidation (FAO) activity in AILI. Egr1 transcriptionally upregulated FAO-related genes in hepatocytes. Notably, the knockdown of acetyl-coenzyme A acyltransferase 2 (Acaa2), a key gene involved in FAO, diminished this protective effect of Egr1. Clinically, EGR1 was markedly increased in liver tissues from AILI patients. Interestingly, EGR1 levels of liver tissues and serum samples were also obviously higher in idiosyncratic DILI patients. Conclusions: Egr1 confers adaptive protection in AILI, mediated via the transcriptional upregulation of Acaa2, which improves mitochondrial FAO, and might be a potential biomarker and novel therapeutic target for AILI.

The Effect of EGR1 on the Proliferation of Dermal Papilla Cells.

Early growth response factor 1 (EGR1) is a zinc-finger transcription factor that plays a vital role in the development of hair follicles. According to our previous studies, EGR1 is a transcriptional promoter of the bone morphogenetic protein 7 (BMP7), a candidate gene involved in the proliferation of dermal papilla cells. Since hair follicles are the basis of lambskin pattern formation and dermal papilla cells (DPCs) act on hair follicle growth, in order to elucidate the role of EGR1 and hair follicles, this study aimed to investigate the biological role of EGR1 in DPCs. In our study, the EGR1 coding sequence (CDS) region was firstly cloned by polymerase chain reaction, and bioinformatics analysis was performed. Then, the function of EGR1 was detected by 5-ethynyl-2'-deoxyuridine (EDU) and Cell Counting Kit-8 (CCK8), and Western blot (WB) was conducted to analyze the cellular effect of EGR1 on DPCs. The proliferative effect of EGR1 on DPCs was also further confirmed by detecting its expression by qPCR and WB on marker genes of proliferation, including PCNA and CDK2. The sequence of the EGR1 CDS region of a lamb was successfully cloned, and its nucleic acid sequence was analyzed and found to be highly homologous to Rattus norvegicus, Mus musculus, Bos taurus and Homo sapiens. Predictive analysis of the protein encoded by EGR1 revealed that it is an extra-membrane protein, and not a secretory protein, with subcellular localization in the nucleus and cytoplasm. The proliferative effect of DPCs was significantly stronger (p < 0.01) in EGR1 up-regulated DPCs compared to the controls, while the opposite result was observed in EGR1 down-regulated DPCs. Markers of proliferation including PCNA and CDK2 also appeared to be differentially upregulated in EGR1 gene overexpression compared to the controls, with the opposite result in EGR1 gene downregulation. In summary, our study revealed that EGR1 promotes the proliferation of DPCs, and we speculate that EGR1 may be closely associated with hair follicle growth and development.

Cyclic mechanical stretch regulates the AMPK/Egr1 pathway in tenocytes via Ca2+-mediated mechanosensing.

PURPOSE: Mechanical stimuli are essential for the maintenance of tendon tissue homeostasis. The study aims to elucidate the mechanobiological mechanisms underlying the maintenance of tenocyte homeostasis by cyclic mechanical stretch under high-glucose (HG) condition. MATERIALS AND METHODS: Primary tenocytes were isolated from rat Achilles tendon and 2D-cultured under HG condition. The in vitro effects of a single bout, 2-h cyclic biaxial stretch session (1 Hz, 8%) on primary rat tenocytes were explored through Flexcell system. Cell viability, tenogenic gene expression, intracellular calcium concentration, focal adhesion kinase (FAK) expression, and signaling pathway activation were analyzed in tenocytes with or without mechanical stretch. RESULTS: Mechanical stretch increased tenocyte proliferation and upregulated early growth response protein 1 (Egr1) expression. An increase in intracellular calcium was observed after 30 min of stretching. Mechanical stretch phosphorylated FAK, calmodulin-dependent protein kinase kinase 2 (CaMKK2), and 5' adenosine monophosphate-activated protein kinase (AMPK) in a time-dependent manner, and these effects were abrogated after blocking intracellular calcium. Inhibition of FAK, CaMKK2, and AMPK downregulated the expression of Egr1. In addition, mechanical stretch reinforced cytoskeletal organization via calcium (Ca2+)/FAK signaling. CONCLUSIONS: Our study demonstrated that mechanical stretch-induced calcium influx activated CaMKK2/AMPK signaling and FAK-cytoskeleton reorganization, thereby promoting the expression of Egr1, which may help maintain tendon cell characteristics and homeostasis in the context of diabetic tendinopathy.

LncRNA-COX2 inhibits Fibroblast Activation and Epidural Fibrosis by Targeting EGR1.

Rationale: Epidural fibrosis is one of the contributors to failed back surgery syndrome (FBSS) with a high incidence of about 80,000 cases per year. The fibrosis spreads from the operative region to the dura mater or the nerve root and results in functional incapacity and pain after laminectomy. Our previous study showed that down-regulation of lncRNA-COX2 is involved in the epidural scar formation. However, it remains unknown whether lncRNA-COX2 participate in the fibroblast activation and epidural fibrogenesis. Methods: LncRNA-COX2 and EGR1 expression were assessed by qRT-PCR and western blotting. Fibroblasts differentiation, proliferation and migration was determined by Collagen I/ɑ-SMA, 5-ethynyl-2'-deoxyuridine (EdU) and Transwell Assay respectively. Luciferase reporter assay was performed for the verification of target of LncRNA-COX2. Laminectomy was performed to establish the model of epidural fibrosis in mice. Epidural scar was evaluated by hematoxylin and eosin (HE) staining and Masson Trichrome staining. Results: Based on the result of transcriptome profiling, we found LncRNA-COX2 was significantly decreased in epidural tissues after laminectomy and in activated fibrotic fibroblasts. In vitro, overexpression of LncRNA-COX2 suppressed epidural fibrogenesis by inhibiting fibroblasts differentiation, proliferation and migration. Mechanistically, LncRNA-COX2 functioned as competing endogenous RNA (ceRNA) of EGR1. Gain of LncRNA-COX2 significantly decreased the expression of EGR1 and showed anti-fibrotic effect while EGR1 was markedly increased after loss of LncRNA-COX2. In vivo, LncRNA-COX2 attenuated laminectomy-induced epidural fibrosis in mice. Conclusion: In summary, the results demonstrated that LncRNA-COX2 showed anti-fibrotic effect by targeting EGR1 and identified LncRNA-COX2 as therapeutic molecule for preventing aberrant epidural fibrosis.

JNK-dependent phosphorylation and nuclear translocation of EGR-1 promotes cardiomyocyte apoptosis.

Myocardial apoptosis induced by myocardial ischemia and hyperlipemia are the main causes of high mortality of cardiovascular diseases. It is not clear whether there is a common mechanism responsible for these two kinds of cardiomyocyte apoptosis. Previous studies demonstrated that early growth response protein 1 (EGR-1) has a pro-apoptotic effect on cardiomyocytes under various stress conditions. Here, we found that EGR-1 is also involved in cardiomyocyte apoptosis induced by both ischemia and high-fat, but how EGR-1 enters the nucleus and whether nuclear EGR-1 (nEGR-1) has a universal effect on cardiomyocyte apoptosis are still unknown. By analyzing the phosphorylation sites and nucleation information of EGR-1, we constructed different mutant plasmids to confirm that the nucleus location of EGR-1 requires Ser501 phosphorylation and regulated by JNK. Furthermore, the pro-apoptotic effect of nEGR-1 was further explored through genetic methods. The results showed that EGR-1 positively regulates the mRNA levels of apoptosis-related proteins (ATF2, CTCF, HAND2, ELK1), which may be the downstream targets of EGR-1 to promote the cardiomyocyte apoptosis. Our research announced the universal pro-apoptotic function of nEGR-1 and explored the mechanism of its nucleus location in cardiomyocytes, providing a new target for the "homotherapy for heteropathy" to cardiovascular diseases.

EGR1 Suppresses Porcine Epidemic Diarrhea Virus Replication by Regulating IRAV To Degrade Viral Nucleocapsid Protein.

Porcine epidemic diarrhea virus (PEDV) is a globally distributed alphacoronavirus that has reemerged lately, resulting in large economic losses. During viral infection, type I interferon (IFN-I) plays a vital role in the antiviral innate immunity. However, PEDV has evolved strategies to limit IFN-I production. To suppress virus replication, the host must activate IFN-stimulated genes and some host restriction factors to circumvent viral replication. This study observed that PEDV infection induced early growth response gene 1 (EGR1) expression in PEDV-permissive cells. EGR1 overexpression remarkably suppressed PEDV replication. In contrast, depletion of EGR1 led to a significant increase in viral replication. EGR1 suppressed PEDV replication by directly binding to the IFN-regulated antiviral (IRAV) promoter and upregulating IRAV expression. A detailed analysis revealed that IRAV interacts and colocalizes with the PEDV nucleocapsid (N) protein, inducing N protein degradation via the E3 ubiquitin ligase MARCH8 to catalyze N protein ubiquitination. Knockdown of endogenous MARCH8 significantly reversed IRAV-mediated N protein degradation. The collective findings demonstrate a new mechanism of EGR1-mediated viral restriction, in which EGR1 upregulates the expression of IRAV to degrade PEDV N protein through MARCH8. IMPORTANCE PEDV is a highly contagious enteric coronavirus that has rapidly emerged worldwide and has caused severe economic losses. No currently available drugs or vaccines can effectively control PEDV. PEDV has evolved many strategies to limit IFN-I production. We identified EGR1 as a novel host restriction factor and demonstrated that EGR1 suppresses PEDV replication by directly binding to the IRAV promoter and upregulating the expression of IRAV, which interacts with and degrades the PEDV N protein via the E3 ubiquitin ligase MARCH8 to catalyze nucleocapsid protein ubiquitination, which adds another layer of complexity to the innate antiviral immunity of this newly identified restriction factor. A better understanding of the innate immune response to PEDV infection will aid the development of novel therapeutic targets and more effective vaccines against virus infection.

Type 2 Diabetes Mellitus Induced Paracrine Effects on Breast Cancer Metastasis Through Extracellular Vesicles Derived from Human Mesenchymal Stem Cells.

Cancer metastasis is the leading cause of mortality among breast cancer patients. Type 2 diabetes mellitus (T2DM) has been suggested as a risk factor of breast cancer; however, whether or not T2DM is associated with breast tumor metastasis remains unclear. In this study, we examined the involvement of T2DM with breast cancer metastasis by a combined approach of a meta-analysis and experimental research. The results of a systematic review and meta-analysis suggested that diabetes significantly increases the risk of lymph node metastasis by 1.10-fold (P < 0.01). Consistently, our data from experimental research showed that T2DM induced paracrine effects of mesenchymal stem cells (MSCs), a key contributor to cancer progression, to stimulate metastasis of breast cancer cells (BCCs) by two independent mechanisms. First, T2DM induced the excess secretion of interleukin 6 (IL6) from MSCs, which activated the JAK/STAT3 pathway in BCCs, thus promoting the metastasis of BCCs. Second, beside the EGR-1-/IL6-dependent mechanism, T2DM altered the functions of MSC-derived extracellular vesicles (EVs), which are highly associated with the metastasis of BCCs. Our present study showed that T2DM is a risk factor for breast cancer metastasis, and MSC-derived EVs might be useful for developing a novel anti-breast cancer therapy strategy.

Novelty enhances memory persistence and remediates propranolol-induced deficit via reconsolidation.

Memory reactivation has been shown to open a time window for memory modulation. The majority of the methodological or pharmacological approaches target disruption of reconsolidation to weaken aversive memories. However, methods to improve appetitive memory persistence through reconsolidation or to reverse drug-induced reconsolidation impairment are limited. To improve memory persistence, previous studies show that a novel event, introduced around the time of memory encoding, enables the persistence of an otherwise decayed memory. This is mainly through a memory consolidation process. The current study first investigated if a novel event introduced during memory reactivation improves memory persistence through reconsolidation. Using a rodent appetitive spatial paradigm, similar to the human everyday experience of recalling where an item is located, a novel event around memory reactivation facilitated the persistence of spatial memory. This facilitation did not occur when the novel event was omitted and the protein synthesis-dependent reconsolidation was not affected by zif268 anti-sense in the dorsal hippocampus. Furthermore, beta-adrenergic antagonists, propranolol, impaired reconsolidation of appetitive spatial memory and contextual fear conditioning. A novel event after memory reactivation could reverse this impairment due to propranolol. Together, this study provides methods and confirmation for improving memory persistence during memory reactivation and reconsolidation.

Egr-1 Maintains NSC Proliferation and Its Overexpression Counteracts Cell Cycle Exit Triggered by the Withdrawal of Epidermal Growth Factor.

In adult mammals, neural stem cells (NSCs) reside in specialized niches at the level of selected CNS regions, such as the subventricular zone (SVZ). The signaling pathways that reg-ulate NSC proliferation and differentiation remain poorly understood. Early growth response protein 1 (Egr-1) is an important transcription factor, widely studied in the adult mammalian brain, mediating the activation of target genes by a variety of extracellular stimuli. In our study, we aimed at testing how Egr-1 regulates adult NSCs derived from mouse SVZ and, in particular, the interplay between Egr-1 and the proliferative factor EGF. We demonstrate that Egr-1 expression in NSCs is induced by growth factor stimulation, and its level decreases after EGF deprivation or by using AG1478, an inhibitor of the EGF/EGFR signaling pathway. We also show that Egr-1 overexpression rescues the cell proliferation decrease observed either after EGF removal or upon treatment with AG1478, suggesting that Egr-1 works downstream of the EGF pathway. To better understand this mechanism, we investigated targets downstream of both the EGF pathway and Egr-1, and found that they regulate genes involved in NSC proliferation, such as cell cycle regulators, cyclins, and cyclin-dependent kinase inhibitors.

131I therapy mediated by sodium/iodide symporter combined with kringle 5 has a synergistic therapeutic effect on glioma.

Glioblastoma (GBM) is the most common and most aggressive primary brain tumor; the prognosis of patients with GBM remains poor. The sodium/iodide symporter (NIS) can be used to absorb several isotopes, such as 131I for nuclear medicine imaging and radionuclide therapy. Previously, we found that the early growth response-1 (Egr1) promoter had an 131I radiation positive feedback effect on the NIS gene. Kringle 5 (K5), a kringle domain of plasminogen, induced endothelial cell apoptosis. We investigated the effect of K5 combined with the 131I radiation positive feedback effect (Egr1-NIS) for treating malignant U87 glioma cells using a lentiviral vector. We successfully constructed a stable U87 glioma cell line, U87-K5-Egr1-NIS. The radio-inducible Egr1 promoter induced an 131I radiation positive feedback effect absorbed by NIS. Mediated by 131I, K5 increased glioma cell apoptosis; 131I radiation also increased endothelial cell sensitivity to K5-induced apoptosis. The combined therapy had a synergistic effect on the antitumor efficacy of glioma treatment, not only increasing tumor cell apoptosis but also significantly inhibiting tumor cell proliferation and reducing capillary density in U87 glioma tissues.

[Steroid drugs and GM-CSF modulates activity of Egr-1 in glioma cells]. / Fármacos esteroides y el GM-CSF modulan la actividad de Egr-1 en células de glioma.

INTRODUCTION: The Egr-1 protein is a transcriptional factor responsive to early growth. Transcriptional regulation of the promoter has been described like responsive to physical stress, osmotic changes, and cellular growth marker. However, there is no report about the pharmacological effect on the transcriptional regulation in gliomas. Hereby we report the modulation of the Egr-1 promoter transcriptional activity induced by the Granulocytes Macrophages Colony Stimulating Factor (GM-CSF) and steroid drugs in human glioma cells (CH235-GM Grade II, U373-GM Grade III, D54-GM Grade IV) using a reporter system transduced by a recombinant adenoviral vector AdEgr-1/luc7. METHODS: Human glioma cells shows with different malignity grade (CH235-GM Grado II; U373-GM Grado III; D54-GM Grado IV) were transduced with no replicative adenoviral vector AdEgr-1/Luc7 and exposed to drugs as progesterone, ß-estradiol and betametasone, and GM-CSF. Transcriptional activity of the egr-1 promoter was quantified by Luciferase reporter gene, cloned downstream to the tata box. Luciferase activity was quantified from whole cell proteins using luminometry assays. RESULTS: U373-GM cell line with GM-CSF, shows an increment on transcriptional activity of Egr-1 promoter, also in endogen way. U373-GM showed a positive regulation of Egr-1, with steroid drugs on the times analyzed. Steroid drugs as progesterone, ß-estradiol and betametasone, shows a pleiotropic behavior on CH235-GM and D54-GM, glioma cell lines. CONCLUSIONS: Inhibition or activation response of Egr-1 promoter shows new framework to explore a mechanism of action of steroid drugs on genetic and epigenetic regulation on tumoral process.

The transcription factors Egr1 and Egr2 have opposing influences on adipocyte differentiation.

The zinc finger-containing transcription factors Egr1 (Krox24) and Egr2 (Krox20) have been implicated in the proliferation and differentiation of many cell types. Egr2 has earlier been shown to play a positive role in adipocyte differentiation, but the function of Egr1 in this context is unknown. We compared the roles of Egr1 and Egr2 in the differentiation of murine 3T3-L1 adipocytes. Egr1 protein was rapidly induced after addition of differentiation cocktail, whereas Egr2 protein initially remained low before increasing on days 1 and 2, concomitant with the disappearance of Egr1. In marked contrast to the effects of Egr2, differentiation was inhibited by ectopic expression of Egr1 and potentiated by knockdown of Egr1. The pro-adipogenic effects of Egr1 knockdown were particularly notable when isobutylmethylxanthine (IBMX) was omitted from the differentiation medium. However, knockdown of Egr1 did not affect CCAAT/enhancer binding protein (C/EBP)beta protein expression or phosphorylation of CREB Ser133. Further, Egr1 did not directly affect the activity of promoters for the master adipogenic transcription factors, C/EBPalpha or peroxisome proliferator-activated receptor-gamma2, as assessed in luciferase reporter assays. These data indicate that Egr1 and Egr2 exert opposing influences on adipocyte differentiation and that the careful regulation of both is required for maintaining appropriate levels of adipogenesis. Further, the pro-differentiation effects of IBMX involve suppression of the inhibitory influence of Egr1.