Egr-1 Target Research Articles

BRD4 inhibitor reduces exhaustion and blocks terminal differentiation in CAR-T cells by modulating BATF and EGR1

BackgroundExhaustion is a key factor that influences the efficacy of chimeric antigen receptor T (CAR-T) cells. Our previous study demonstrated that a bromodomain protein 4 (BRD4) inhibitor can revise the phenotype and function of exhausted T cells from leukemia patients. This study aims to elucidate the mechanism by which a BRD4 inhibitor reduces CAR-T cell exhaustion using single-cell RNA sequencing (scRNA-Seq).MethodsExhausted CD123-specific CAR-T cells were prepared by co-culture with CD123 antigen-positive MV411 cells. After elimination of MV411 cells and upregulation of inhibitory receptors on the surface, exhausted CAR-T cells were treated with a BRD4 inhibitor (JQ1) for 72 h. The CAR-T cells were subsequently isolated, and scRNA-Seq was conducted to characterize phenotypic and functional changes in JQ1-treated cells.ResultsBoth the proportion of exhausted CD8+ CAR-T cells and the exhausted score of CAR-T cells decreased in JQ1-treated compared with control-treated cells. Moreover, JQ1 treatment led to a higher proportion of naïve, memory, and progenitor exhausted CD8+ CAR-T cells as opposed to terminal exhausted CD8+ CAR-T cells accompanied by enhanced proliferation, differentiation, and activation capacities. Additionally, with JQ1 treatment, BATF activity and expression in naïve, memory, and progenitor exhausted CD8+ CAR-T cells decreased, whereas EGR1 activity and expression increased. Interestingly, AML patients with higher EGR1 and EGR1 target gene ssGSEA scores, coupled with lower BATF and BATF target gene ssGSEA scores, had the best prognosis.ConclusionsOur study reveals that a BRD4 inhibitor can reduce CAR-T cell exhaustion and block exhausted T cell terminal differentiation by downregulating BATF activity and expression together with upregulating EGR1 activity and expression, presenting an approach for improving the effectiveness of CAR-T cell therapy.

Toe1 promotes proliferation and differentiation of neural progenitor cells

Toe1 (target of EGR1, member 1) is a 3′- exonuclease in the deadenylase family, essential for the maturation of small nuclear RNAs. Mutations in Toe1 are linked to pontocerebellar hypoplasia 7 (PCH7), a severe neurodegenerative syndrome affecting infants, characterized by progressive neurodegeneration, developmental delay, and genital abnormalities. The pathogenic mechanisms of PCH7 are unclear but are thought to involve abnormal neural stem cell (NSC) development during embryogenesis. This study investigates Toe1's role in NSC development using the C17.2 NSC line. Colony formation, EdU incorporation, and CFSE staining assays showed that Toe1 knockout inhibited C17.2 cell proliferation. Upon inducing differentiation, Toe1 knockout significantly reduced cell dendrites. Immunofluorescence, qPCR, and Western blot analyses indicated that Toe1 knockout suppressed the expression of neuronal marker βIII-tubulin and glial cell marker Gfap, thereby inhibiting C17.2 cell differentiation. Additionally, Toe1 knockout reduced the expression of Dll1 and Jag1, suggesting an inhibition of Notch signaling. High-throughput transcriptome sequencing revealed that Toe1 influenced calcium ion binding, ECM, and amino acid catabolism in undifferentiated C17.2 cells, and peptidase activity, chemotactic factors, ECM, and TNF signaling in differentiated cells. These findings underscore Toe1's critical regulatory role in NSC proliferation and differentiation, with significant implications for developing therapeutic targets for neurodegenerative diseases such as PCH7.

Inhibition of Transverse Aortic Constriction Induced Myocardial Hypertrophy by Knocking out the Target of the Early Growth Response-1 to Inhibit Apoptosis and Autophagy

Myocardial hypertrophy, a significant contributor to the development of heart failure, continues to be prevalent. Early growth response-1 (EGR-1) is closely linked to the development of diverse myocardial conditions. The target of EGR1 (TOE1) is a critical factor in myocardial hypertrophy, but its regulatory function remains unclear. Myocardial cell injury was induced by angiotensin II. TOE1 knockout mice and cells were generated to investigate its impact on myocardial hypertrophy. TUNEL staining was employed to assess cell apoptosis. Furthermore, western blotting and qRT-PCR were performed to measure the expression of target genes. The results revealed that knockout of TOE1 effectively inhibited myocardial hypertrophy and injury caused by transverse aortic constriction. In vivo experiments demonstrated that TOE1 knockout improved myocardial function and suppressed inflammatory factors, oxidative stress, apoptosis, and autophagy levels. In vitro, TOE1 knockout suppressed cell apoptosis, mitochondrial damage, and the intensity of reactive oxygen species. Additionally, it inhibited the expression of apoptosis- and autophagy-related genes. These findings introduce a promising avenue for preventing and treating myocardial hypertrophy.

Novel compound heterozygous missense variants in TOE1 gene associated with pontocerebellar hypoplasia type 7

BackgroundPontocerebellar hypoplasia type 7(PCH7)is a neurodegenerative disease related to autosomal recessive variants in the target of EGR1 (TOE1)gene. Biallelic mutation in the TOE1 gene causes global developmental delay, cognitive and psychomotor impairment, hypotonia, breathing abnormalities, and gonadal abnormalities. This study examined the clinical and genetic features of a 2-year-old patient carrying novel compound heterozygous variants in the TOE1 gene, mutations of previously reported 14 PCH7 patients were reviewed. MethodsClinical data of the 2-year-old patient were captured. Trio- whole exome sequencing (Trio-WES) was performed to identify pathogenic variants. Sanger sequencing was further used to verify the variants. In silico analysis was performed to explain the pathogenicity. ResultsHerein, we described the clinical features of the 2-year-old patient diagnosed with PCH7 caused by mutations in the TOE1gene. The kid was presenting with global development delay and gonadal abnormalities. Brain imaging revealed hypoplasia of the cerebellum and pons with ambiguous genitalia. Trio-WES revealed novel compound heterozygous missense variants in TOE1gene (c.911C > T p.S304L, c.161C > T p.A54V). Multiple in silico tools predicted the deleterious effects of the mutations. ConclusionThe novel compound heterozygous missense mutation in the TOE1 gene identified in the proband broadened the genotypic and phenotypic spectrum of disorders associated with PCH7. Our findings provide critical information for the differential diagnosis of rare neurodevelopment disorders and genetic counselling.

Alteration in DNA-binding affinity of Wilms tumor 1 protein due to WT1 genetic variants associated with steroid - resistant nephrotic syndrome in children

Approximately one third of children with steroid-resistant nephrotic syndrome (SRNS) carry pathogenic variants in one of the many associated genes. The WT1 gene coding for the WT1 transcription factor is among the most frequently affected genes. Cases from the Czech national SRNS database were sequenced for exons 8 and 9 of the WT1 gene. Eight distinct exonic WT1 variants in nine children were found. Three children presented with isolated SRNS, while the other six manifested with additional features. To analyze the impact of WT1 genetic variants, wild type and mutant WT1 proteins were prepared and the DNA-binding affinity of these proteins to the target EGR1 sequence was measured by microscale thermophoresis. Three WT1 mutants showed significantly decreased DNA-binding affinity (p.Arg439Pro, p.His450Arg and p.Arg463Ter), another three mutants showed significantly increased binding affinity (p.Gln447Pro, p.Asp469Asn and p.His474Arg), and the two remaining mutants (p.Cys433Tyr and p.Arg467Trp) showed no change of DNA-binding affinity. The protein products of WT1 pathogenic variants had variable DNA-binding affinity, and no clear correlation with the clinical symptoms of the patients. Further research is needed to clarify the mechanisms of action of the distinct WT1 mutants; this could potentially lead to individualized treatment of a so far unfavourable disease.

A DNA Nanocomplex Containing Cascade DNAzymes and Promoter-Like Zn-Mn-Ferrite for Combined Gene/Chemo-dynamic Therapy.

Sequential control of exogenous chemical events inside cells is a promising way to regulate cell functions and fate. Herein we report a DNA nanocomplex containing cascade DNAzymes and promoter-like Zn-Mn-Ferrite (ZMF), achieving combined gene/chemo-dynamic therapy. The promoter-like ZMF decomposed in response to intratumoral glutathione to release a sufficient quantity of metal ions, thus promoting cascade DNA/RNA cleavage and free radical generation. Two kinds of DNAzymes were designed for sequential cascade enzymatic reaction, in which metal ions functioned as cofactors. The primary DNAzyme self-cleaved the DNA chain with Zn2+ as cofactor, and produced the secondary DNAzyme; the secondary DNAzyme afterwards cleaved the EGR-1 mRNA, and thus downregulated the expression of target EGR-1 protein, achieving DNAzyme-based gene therapy. Meanwhile, the released Zn2+ , Mn2+ and Fe2+ induced Fenton/Fenton-like reactions, during which free radicals were catalytically generated and efficient chemo-dynamic therapy was achieved. In a breast cancer mouse model, the administration of DNA nanocomplex led to a significant therapeutic efficacy of tumor growth suppression.

Gfi1-Mediated Repression of C-Fos, Egr1 and Egr2, and Inhibition of ERK1/2 Signaling Contribute to the Role of Gfi1 in Granulopoiesis

Gfi1 is a transcriptional repressor that plays an important role in hematopoiesis. In the myeloid system, Gfi1 promotes granulopoiesis and antagonizes the alternative monocyte development in part through repressing PU.1 and CSF1 , which promote monopoiesis. However, the molecular mechanism by which Gfi1 favors neutrophil over monocyte development remains incompletely understood. G-CSF is a lineage-specific cytokine that plays a dominant role in granulopoiesis. We recently showed that a G-CSFR mutant in which tyrosine 729 was mutated to phenylalanine (Y729F) promoted monocyte rather than neutrophil development in myeloid precursors, which was associated with prolonged activation of ERK1/2 and augmented activation of downstream targets c-Fos and Egr1. To address whether Gfi1 may promote granulopoiesis independent of its repression of Csf1 , we established murine myeloid 32D and multipotent FDCP-mix cells that constitutively expressed G-CSFR Y729F and inducibly expressed Gfi1 in response to doxycycline (Dox; 32D/Y729F/Gfi1 and FDCP/Y729F/Gfi1). 32D and FDCP-mix cells showed no response to CSF1. In the absence of Dox, 32D/Y729F/Gfi1 and FDCP/Y729F/Gfi1 differentiated into monocytes when cultured in G-CSF. Interestingly, addition of Dox to induce Gfi1 expression rescued neutrophil development in both cell lines. Induction of Gfi1 expression with Dox also markedly attenuated the induction of c-Fos, Egr1 and Egr2 expression in response to G-CSF. In contrast, the mRNA levels of c-Fos, Egr1 and Egr2 were dramatically elevated in lineage marker negative (Lin-) bone marrow (BM) cells from Gfi1-/- mice as compared to BM cells from Gfi1+/+ mice. Chromatin immunoprecipitation (ChIP) and luciferase reporter assays demonstrated that Gfi1 bound to and repressed the activities of c-Fos , Egr1 and Egr2 promoters. c-Fos , Egr1 and Egr2 are immediate early genes that are activated by the ERK1/2 pathway. Notably, G-CSF-stimulated activation of ERK1/2 was stronger in Lin- BM cells from Gfi1-/- mice than in cells from Gfi1+/+ mice. It has been shown that BM cells from Gfi1-/- mice are unable to differentiate into mature neutrophils in vitro , but instead give rise to atypical monocytes. Interestingly, the MEK1/2 inhibitor U0126 or PD0325901 reduced the mRNA levels of c-Fos, Egr1 and Egr2, and partially rescued G-CSF-induced neutrophil development in Gfi1-/- BM cells. Together, our data indicate that Gfi1 inhibits the expression of c-Fos, Egr1 and Egr2 through direct transcriptional repression of their genes and indirect inhibition of ERK1/2 signaling pathway, and that Gfi1-mediated downregulation of c-Fos, Egr1 and Egr2 may contribute to the role of Gfi1 in granulopoiesis. DisclosuresNo relevant conflicts of interest to declare.

The Crucial Role of PPARγ-Egr-1-Pro-Inflammatory Mediators Axis in IgG Immune Complex-Induced Acute Lung Injury.

BackgroundThe ligand-activated transcription factor peroxisome proliferator-activated receptor (PPAR) γ plays crucial roles in diverse biological processes including cellular metabolism, differentiation, development, and immune response. However, during IgG immune complex (IgG-IC)-induced acute lung inflammation, its expression and function in the pulmonary tissue remains unknown.ObjectivesThe study is designed to determine the effect of PPARγ on IgG-IC-triggered acute lung inflammation, and the underlying mechanisms, which might provide theoretical basis for therapy of acute lung inflammation.SettingDepartment of Pathogenic Biology and Immunology, Medical School of Southeast UniversitySubjectsMice with down-regulated/up-regulated PPARγ activity or down-regulation of Early growth response protein 1 (Egr-1) expression, and the corresponding controls.InterventionsAcute lung inflammation is induced in the mice by airway deposition of IgG-IC. Activation of PPARγ is achieved by using its agonist Rosiglitazone or adenoviral vectors that could mediate overexpression of PPARγ. PPARγ activity is suppressed by application of its antagonist GW9662 or shRNA. Egr-1 expression is down-regulated by using the gene specific shRNA.Measures and Main ResultsWe find that during IgG-IC-induced acute lung inflammation, PPARγ expression at both RNA and protein levels is repressed, which is consistent with the results obtained from macrophages treated with IgG-IC. Furthermore, both in vivo and in vitro data show that PPARγ activation reduces IgG-IC-mediated pro-inflammatory mediators’ production, thereby alleviating lung injury. In terms of mechanism, we observe that the generation of Egr-1 elicited by IgG-IC is inhibited by PPARγ. As an important transcription factor, Egr-1 transcription is substantially increased by IgG-IC in both in vivo and in vitro studies, leading to augmented protein expression, thus amplifying IgG-IC-triggered expressions of inflammatory factors via association with their promoters.ConclusionDuring IgG-IC-stimulated acute lung inflammation, PPARγ activation can relieve the inflammatory response by suppressing the expression of its downstream target Egr-1 that directly binds to the promoter regions of several inflammation-associated genes. Therefore, regulation of PPARγ-Egr-1-pro-inflammatory mediators axis by PPARγ agonist Rosiglitazone may represent a novel strategy for blockade of acute lung injury.

Using CRISPR/Cas9 to Investigate the Role of Egr1 and NAB2 in Neuronal Differentiation of PC12 Cells

PC12 cells are a common model system used to study the molecular events underlying neuronal differentiation due to their ability to differentiate into neuronal cells following treatment with nerve growth factor (NGF). NGF induces neuronal differentiation through activation of the Ras/Raf/MEK/ERK signaling pathway, which in turn activates a transcriptional program leading to up‐regulation of neuronal genes, neurite outgrowth, and establish electrical excitability. Interestingly, treatment with epidermal growth factor (EGF) also activates Ras/Raf/MEK/ERK signaling, but induces proliferation rather than differentiation. The differential effects of NGF versus EGF are due to differences in ERK signal duration; EGF induces more transient ERK signaling that lasts 30–60 min, whereas NGF induces more sustained ERK signaling that lasts 4–6 h. Work in our lab and others indicate that sustained ERK signaling in response to NGF drives differentiation in part through sustained activity of the transcription factor Egr1 and, in turn, sustained expression of Egr1 target genes. Egr1 activity in response to EGF and NGF is regulated at two levels. First, Egr1 levels rapidly rise to peak levels by 1 h after treatment and then decline to baseline levels; similar to ERK signaling, Egr1 levels are sustained longer in response to NGF versus EGF. Second, one Egr1 target gene is its corepressor NAB2, which binds and represses Egr1 transactivation once expressed. This project was designed to further test our hypothesis that sustained Egr1 activity is a major driver of PC12 neuronal differentiation by evaluating whether knockout of NAB2 will lead to sustained Egr1 activity in response to EGF and, in turn, neuronal differentiation. To acquire PC12 cells that lack NAB2 protein, we used CRISPR/Cas9 technology to knockout the NAB2 gene. More specifically, a CRISPR NAB2‐KO plasmid was purchased from Santa Cruz Biotechnology, which encodes a green fluorescent protein (GFP) selectable marker, the Cas9 protein, and the guide RNA (gRNA). The plasmid had three variations for the gRNA which target different locations in the NAB2 gene. One day after transfection, GFP‐positive cells were sorted into 96‐well plates to isolate clones. Following clonal expansion, clones were first screened for NAB2 expression by Western blot, which identified eight putative knockout clones. Genomic DNA was then extracted from the eight clones, the NAB2 gene amplified by PCR, and the PCR project then subjected to Sanger sequencing to identify the INDELs responsible for NAB2 knockout. Ongoing work is examining the effects of NAB2 knockout on Egr1 levels and activity in response to EGF and NGF, as well as neuronal differentiation based on neurite outgrowth with the prediction that NAB2 knockout will result in sustained Egr1 activity and neuronal differentiation in response to both growth factors.Support or Funding InformationBridgewater State University Undergraduate Research Program

NAB2-STAT6 fusion protein mediates cell proliferation and oncogenic progression via EGR-1 regulation

Solitary fibrous tumors are rare mesenchymal tumors derived from soft tissues and vascular walls. NAB2-STAT6 fusion gene serves as a marker gene for this disease and consists of the truncated repressor domain of NGFI-A-Binding protein 2 (NAB2) and the intact activation domain of STAT6. In this study, we found that EGR-1 and the proliferation-related EGR-1 target gene IGF2 were upregulated in NIH-3T3 cells transfected with NAB2-STAT6. Additionally, p-Rb (Ser795) and cyclin D1 levels were upregulated, and cell proliferation was also enhanced. We identified that treatment with the IGF2 inhibitor reduced cell proliferation in NIH-3T3 cells transfected with NAB2-STAT6. The oncogenic progression was enhanced in NIH-3T3 cells transfected with NAB2-STAT6 compared with those transfected with the empty vector. Taken together, our study suggests that the NAB2-STAT6 fusion gene is associated with cell proliferation through EGR-1 transcriptional expression and IGF2 can be a drug target for the treatment of solitary fibrous tumors.

Design and Structure Determination of a Composite Zinc Finger Containing a Nonpeptide Foldamer Helical Domain.

A number of foldamer backbones have been described as useful mimics of protein secondary structure elements, enabling for example the design of synthetic oligomers with the ability to engage specific protein surfaces. Synthetic folded backbones can also be used to create artificial proteins in which a folded peptide segment (e.g., an α-helix, a loop) is replaced by its unnatural counterpart, with the expectation that the resulting molecule would maintain its ability to fold while manifesting new exploitable features. The similarities in screw sense, pitch, and polarity between peptide α-helices and oligourea 2.5-helices suggest that a tertiary structure could be retained when swapping the two backbones in a protein sequence. In the present work, we move a step toward the creation of such composite proteins by replacing the 10-residue long original α-helical segment in the Cys2His2 zinc finger 3 of transcription factor Egr1 (also known as Zif268) by an oligourea sequence bearing two appropriately spaced imidazole side chains for zinc coordination. We show by spectroscopic techniques and mass spectrometry analysis under native conditions that the ability of the peptide/oligourea hybrid to coordinate the zinc ion is not affected by the foldamer replacement. Moreover, detailed NMR analysis provides evidence that the engineered zinc finger motif adopts a folded structure in which the native β-sheet arrangement of the peptide region and global arrangement of DNA-binding side chains are preserved. Titration in the presence of the Egr1 target DNA sequence supports binding to GC bases as reported for the wild-type zinc finger.

Leptin is a direct transcriptional target of EGR1 in human breast cancer cells.

Leptin is a cytokine that regulates energy metabolism. Leptin can promote breast cancer progression in obese women. However, the mechanism of regulation of leptin expression in breast cancer cells is unclear. Tumor necrosis factor-alpha (TNF-α) stimulated the transcription of the leptin gene. Using mutant promoter constructs, we demonstrated that the EGR1-binding motif in the proximal region of the leptin gene is required for leptin transcription by TNF-α. Forced expression of EGR1 stimulated leptin promoter activity, whereas silencing of EGR1 by RNA interference reduced TNF-α-induced leptin protein accumulation. The ERK1/2 pathway contributed to the expression of EGR1 and leptin by TNF-α. Our results suggest that EGR1 targets the leptin gene in response to TNF-α stimulation in breast cancer cells.

TOE1 acts as a 3' exonuclease for telomerase RNA and regulates telomere maintenance.

In human cells, telomeres are elongated by the telomerase complex that contains the reverse transcriptase hTERT and RNA template TERC/hTR. Poly(A)-specific ribonuclease (PARN) is known to trim hTR precursors by removing poly(A) tails. However, the precise mechanism of hTR 3′ maturation remains largely unknown. Target of Egr1 (TOE1) is an Asp-Glu-Asp-Asp (DEDD) domain containing deadenylase that is mutated in the human disease Pontocerebella Hypoplasia Type 7 (PCH7) and implicated in snRNA and hTR processing. We have previously found TOE1 to localize specifically in Cajal bodies, where telomerase RNP complex assembly takes place. In this study, we showed that TOE1 could interact with hTR and the telomerase complex. TOE1-deficient cells accumulated hTR precursors, including oligoadenylated and 3′-extended forms, which was accompanied by impaired telomerase activity and shortened telomeres. Telomerase activity in TOE1-deficient cells could be rescued by wild-type TOE1 but not the catalytically inactive mutant. Our results suggest that hTR 3′ end processing likely involves multiple exonucleases that work in parallel and/or sequentially, where TOE1 may function non-redundantly as a 3′-to-5′ exonuclease in conjunction with PARN. Our study highlights a mechanistic link between TOE1 mutation, improper hTR processing and telomere dysfunction in diseases such as PCH7.

Egr-1 transactivates WNT5A gene expression to inhibit glucose-induced β-cell proliferation

Selective β-cell loss is a characteristic of type 2 diabetes mellitus (T2DM). Inhibition of glucose-stimulated β-cell proliferation is one of the in vivo results of the lipotoxicity of saturated fatty acids (SFAs). However, the mechanism by which lipotoxicity inhibits β-cell proliferation is still unclear. In this study, we found palmitate, a saturated fatty acid, inhibited the β-cell proliferation induced by high glucose through the induction of Wnt5a expression in vitro and in vivo. We also found that Wnt5a was both sufficient and necessary for inhibition of β-cell proliferation. Additionally, Egr-1, but not NF-κB, FOXO1, Smad2, Smad3, SP1 or SP3 mediated the expression of Wnt5a. Deletion and site-directed mutagenesis of the WNT5A promoter revealed that activation of WNT5A gene transcription depends primarily on a putative Egr-binding sequence between nucleotides −52 to −44, upstream of the transcription start site. Furthermore, Egr-1 bound directly to this sequence in response to palmitate treatment, both in vitro and in vivo. Moreover, after mice islets were treated with Egr inhibitors, the expression of Wnt5a decreased significantly and the glucose-induced β-cell proliferation inhibited by palmitate was resumed. These findings establish Wnt5a as an Egr-1 target gene in β-cells, uncovering a novel Egr-1/Wnt5a pathway by which saturated free fatty acids block glucose-induced β-cell proliferation. Our study lends support for the potential of Egr-1 inhibitors or Wnt5a antibodies as therapeutics for the treatment of T2DM.

Hyperglycemia induced early growth response-1 regulates vascular dysfunction in human retinal endothelial cells

Early growth response-1 (Egr-1) protein upregulation is reported in diabetes and vascular disorders. This study aims at deciphering its role in hyperglycemia induced changes of retinal endothelium. Human retinal endothelial cells (hRECs) were exposed to hyperglycemia (25mM) and normoglycemia (5.5mM). Gene silencing was done using siRNA against Egr-1. Transcript and protein level analysis of Egr-1 and gene targets were done using qPCR and immunoblotting respectively in hRECs, diabetic and nondiabetic human retina and immunofluorescence for localization in retinal sections. Hyperglycemia induced Egr-1 and vascular endothelial growth factor-A (VEGF-A) but not pigment epithelium derived factor (PEDF) in hRECs. Expression of Egr-1 repressor NGFI-A binding protein-2 (NAB-2) was unaltered. Egr-1 downstream gene targets, tissue factor (TF) and intercellular adhesion molecule-1 (ICAM-1) expression were increased in hRECs which was reduced by Egr-1 silencing in hyperglycemia. Diabetic retina, showed an increase in Egr-1, VEGF-A and gene target TF, ICAM-1 but not NAB-2 and PEDF similar to the changes seen in hyperglycemic hRECs. Hyperglycemic induction of Egr-1 and absence of NAB-2 repression in retinal endothelium, up-regulates downstream genes involved in pro-thrombotic and pro-inflammatory pathways linking Egr-1 in diabetes mediated vascular aberration of retina.

The p16/miR-217/EGR1 pathway modulates age-related tenogenic differentiation in tendon stem/progenitor cells.

Previous studies have shown that the differentiation potential declines with the age of progenitor cells and is linked to altered levels of senescence markers. The purpose of this study was to test whether senescence marker p16 affects age-related tenogenic differentiation in tendon stem/progenitor cells (TSPCs). Young and aged TSPCs were isolated from young/healthy and aged/degenerated human Achilles tendons, respectively. Cellular aging and capacity for tenogenic differentiation were examined. The results showed that the tenogenic differentiation capacity of TSPCs significantly decreases with advancing age. TSPCs from elderly donors showed upregulation of senescence-associated β-galactosidase and p16 and concurrently a decrease in Type I collagen concentration and in the expressions of tendon-related markers: Scx, Tnmd, Bgn, Dcn, Col1, and Col3. Overexpression of p16 significantly inhibited tenogenic differentiation of young TSPCs. Analysis of the mechanism revealed that this effect is mediated by microRNA-217 and its target EGR1. These results indicated that p16 inhibits tenogenic differentiation of TSPCs via microRNA signaling pathways, which may serve as a potential target for the prevention or treatment in the future.

Epithelial Cholesterol Deficiency Attenuates Human Antigen R-linked Pro-inflammatory Stimulation via an SREBP2-linked Circuit

Patients with chronic intestinal ulcerative diseases, such as inflammatory bowel disease, tend to exhibit abnormal lipid profiles, which may affect the gut epithelial integrity. We hypothesized that epithelial cholesterol depletion may trigger inflammation-checking machinery via cholesterol sentinel signaling molecules whose disruption in patients may aggravate inflammation and disease progression. In the present study, sterol regulatory element-binding protein 2 (SREBP2) as the cholesterol sentinel was assessed for its involvement in the epithelial inflammatory responses in cholesterol-depleted enterocytes. Patients and experimental animals with intestinal ulcerative injuries showed suppression in epithelial SREBP2. Moreover, SREBP2-deficient enterocytes showed enhanced pro-inflammatory signals in response to inflammatory insults, indicating regulatory roles of SREBP2 in gut epithelial inflammation. However, epithelial cholesterol depletion transiently induced pro-inflammatory chemokine expression regardless of the well known pro-inflammatory nuclear factor-κB signals. In contrast, cholesterol depletion also exerts regulatory actions to maintain epithelial homeostasis against excessive inflammation via SREBP2-associated signals in a negative feedback loop. Mechanistically, SREBP2 and its induced target EGR-1 were positively involved in induction of peroxisome proliferator-activated receptor γ (PPARγ), a representative anti-inflammatory transcription factor. As a crucial target of the SREBP2-EGR-1-PPARγ-associated signaling pathways, the mRNA stabilizer, human antigen R (HuR) was retained in nuclei, leading to reduced stability of pro-inflammatory chemokine transcripts. This mechanistic investigation provides clinical insights into protective roles of the epithelial cholesterol deficiency against excessive inflammatory responses via the SREBP2-HuR circuit, although the deficiency triggers transient pro-inflammatory signals.

Elk-3 Contributes to the Progression of Liver Fibrosis by Regulating the Epithelial-Mesenchymal Transition.

Background/AimsThe role of Elk-3 in the epithelial-mesenchymal transition (EMT) during liver fibrogenesis remains unclear. Here, we determined the expression of Elk-3 in in vitro and in vivo models and in human liver fibrotic tissues. We also investigated the molecular relationships among Elk-3, early growth response-1 (Egr-1), and the mitogen activated protein kinases (MAPK) pathway during EMT in hepatocytes.MethodsWe established anin vitro EMT model in which normal mouse hepatocyte cell lines were treated with transforming growth factor (TGF)-β1 and a CCl4-induced liver fibrosis model. Characteristics of EMT were determined by evaluating the expression levels of related markers. The expression of Elk-3 and its target Egr-1 were analyzed using Western blotting. Gene silencing of Elk-3 was performed using an siRNA knockdown system.ResultsThe expression levels of mesenchymal markers were increased during TGF-β1-induced EMT of hepatocytes. The expression levels of Elk-3 and Egr-1 were significantly (p<0.05) increased during the EMT of hepatocytes, in CCl4-induced mouse liver fibrotic tissues, and in human liver cirrhotic tissues. Silencing of Elk-3 and inhibition of the Ras-Elk-3 pathway with an inhibitor suppressed the expression of EMT-related markers. Moreover, Elk-3 expression was regulated by p38 MAPK phosphorylation during EMT.ConclusionsElk-3 contributes to the progression of liver fibrosis by modulating the EMT via the regulation of Egr-1 under MAPK signaling.

Role of Erk1/2 Signaling in the Regulation of Neutrophil Versus Monocyte Development in Response to G-CSF and M-CSF

Lineage specification in the hematopoietic system depends on the expression of lineage specific transcription factors. However, the role of hematopoietic cytokines in this process has been controversial and little is known about the intracellular signaling mechanisms by which cytokines instruct lineage choice. G-CSF and M-CSF are two lineage-specific cytokines that play a dominant role in granulopoiesis and monopoiesis, respectively. We show here that a G-CSFR mutant in which tyrosine 729 had been mutated to phenylalanine (Y729F) promoted monocyte rather than neutrophil development in myeloid precursors, which was associated with prolonged activation of Erk1/2 and augmented activation of downstream targets c-Fos and Egr1. Inhibition of Erk1/2 activation or knockdown of c-Fos or Egr1 largely rescued neutrophil development in cells expressing G-CSFR Y729F. We also show that M-CSF, but not G-CSF, stimulated strong and sustained activation of Erk1/2 in mouse lineage marker negative (Lin(-)) bone marrow cells. Significantly, inhibition of Erk1/2 signaling in these cells favored neutrophil over monocyte development in response to M-CSF. Thus, prolonged Erk1/2 activation resulted in monocyte development following G-CSF induction whereas inhibition of Erk1/2 signaling promoted neutrophil development at the expense of monocyte formation in response to M-CSF. These results reveal an important mechanism by which G-CSF and M-CSF instruct neutrophil versus monocyte lineage choice, i.e. differential activation of Erk1/2 pathway.

TOE1 is an inhibitor of HIV-1 replication with cell-penetrating capability

Target of Egr1 (TOE1) is a nuclear protein localized primarily in nucleoli and Cajal bodies that was identified as a downstream target of the immediate early gene Egr1. TOE1 displays a functional deadenylation domain and has been shown to participate in spliceosome assembly. We report here that TOE1 can function as an inhibitor of HIV-1 replication and show evidence that supports a direct interaction of TOE1 with the viral specific transactivator response element as part of the inhibitory mechanism. In addition, we show that TOE1 can be secreted by activated CD8(+) T lymphocytes and can be cleaved by the serine protease granzyme B, one of the main components of cytotoxic granules. Both full-length and cleaved TOE1 can spontaneously cross the plasma membrane and penetrate cells in culture, retaining HIV-1 inhibitory activity. Antiviral potency of TOE1 and its cell-penetrating capability have been identified to lie within a 35-amino-acid region containing the nuclear localization sequence.