Role of PAR1-Egr1 in the Initiation of Thoracic Aortic Aneurysm in Fbln4-Deficient Mice.

OBJECTIVE: Remodeling of the extracellular matrix plays a vital role in cardiovascular diseases. Using a mouse model of postnatal ascending aortic aneurysms (termed Fbln4SMKO), we have reported that abnormal mechanosensing led to aneurysm formation in Fbln4SMKO with an upregulation of the mechanosensitive transcription factor, Egr1 (Early growth response 1). However, the role of Egr1 and its upstream regulator(s) in the initiation of aneurysm development and their relationship to an aneurysmal microenvironment are unknown. Approach and Results: To investigate the contribution of Egr1 in the aneurysm development, we deleted Egr1 in Fbln4SMKO mice and generated double knockout mice (DKO, Fbln4SMKO; Egr1-/-). Aneurysms were prevented in DKO mice (42.8%) and Fbln4SMKO; Egr1+/- mice (26%). Ingenuity Pathway Analysis identified PAR1 (protease-activated receptor 1) as a potential Egr1 upstream gene. Protein and transcript levels of PAR1 were highly increased in Fbln4SMKO aortas at postnatal day 1 before aneurysm formed, together with active thrombin and MMP (matrix metalloproteinase)-9, both of which serve as a PAR1 activator. Concordantly, protein levels of PAR1, Egr1, and thrombin were significantly increased in human thoracic aortic aneurysms. In vitro cyclic stretch assays (1.0 Hz, 20% strain, 8 hours) using mouse primary vascular smooth muscle cells induced marked expression of PAR1 and secretion of prothrombin in response to mechanical stretch. Thrombin was sufficient to induce Egr1 expression in a PAR1-dependent manner. CONCLUSIONS: We propose that thrombin, MMP-9, and mechanical stimuli in the Fbln4SMKO aorta activate PAR1, leading to the upregulation of Egr1 and initiation of ascending aortic aneurysms.

Deciphering the functional role of EGR1 in Prostaglandin F2 alpha induced luteal regression applying CRISPR in corpus luteum of buffalo.

BACKGROUND: PGF2α is essential for the induction of the corpus luteum regression which in turn reduces progesterone production. Early growth response (EGR) proteins are Cys2-His2-type zinc-finger transcription factor that are strongly linked to cellular proliferation, survival and apoptosis. Rapid elevation of EGR1 was observed after luteolytic dose of PGF2α. EGR1 is involved in the transactivation of many genes, including TGFß1, which plays an important role during luteal regression. METHODS: The current study was conducted in buffalo luteal cells with the aim to better understand the role of EGR1 in transactivation of TGFß1 during PGF2α induced luteal regression. Luteal cells from mid stage corpus luteum of buffalo were cultured and treated with different doses of PGF2α for different time durations. Relative expression of mRNAs encoding for enzymes within the progesterone biosynthetic pathway (3ßHSD, CYP11A1 and StAR); Caspase 3; AKT were analyzed to confirm the occurrence of luteolytic event. To determine if EGR1 is involved in the PGF2α induced luteal regression via induction of TGFß1 expression, we knocked out the EGR1 gene by using CRISPR/Cas9. RESULT: The present experiment determined whether EGR1 protein expression in luteal cells was responsive to PGF2α treatment. Quantification of EGR1 and TGFß1 mRNA showed significant up regulation in luteal cells of buffalo at 12 h post PGF2α induction. In order to validate the role of PGF2α on stimulating the expression of TGFß1 by an EGR1 dependent mechanism we knocked out EGR1. The EGR1 ablated luteal cells were stimulated with PGF2α and it was observed that EGR1 KO did not modulate the PGF2α induced expression of TGFß1. In PGF2α treated EGR1 KO luteal cell, the mRNA expression of Caspase 3 was significantly increased compared to PGF2α treated wild type luteal cells maintained for 12 h. We also studied the influence of EGR1 on steroidogenesis. The EGR1 KO luteal cells with PGF2α treatment showed no substantial difference either in the progesterone concentration or in StAR mRNA expression with PGF2α-treated wild type luteal cells. CONCLUSION: These results suggest that EGR1 signaling is not the only factor which plays a role in the regulation of PGF2α induced TGFß1 signaling for luteolysis.

Transcriptional Regulation of GDF15 by EGR1 Promotes Head and Neck Cancer Progression through a Positive Feedback Loop.

Growth and differentiation factor 15 (GDF15), a divergent member of the transforming growth factor-ß (TGF-ß) superfamily, has been reported to be overexpressed in different kinds of cancer types. However, the function and mechanism of GDF15 in head and neck cancer (HNC) remains unclear. The Cancer Genome Atlas (TCGA) data show that the expression of GDF15 is significantly associated with tumor AJCC stage, lymph vascular invasion and tumor grade in HNC. In this study, we confirmed that knockdown of GDF15 attenuated: cell proliferation, migration and invasion via regulation of EMT through a canonical pathway; SMAD2/3 and noncanonical pathways; PI3K/AKT and MEK/ERK in HNC cell lines. Furthermore, we found that early growth response 1 (EGR1) was a transcription factor of GDF15. Interestingly, we also demonstrated that GDF15 could regulate the expression of EGR1, which meant a positive feedback loop occurred between these two factors. Moreover, combined inhibition of both GDF15 and EGR1 in a HNC mouse xenograft model showed significantly decreased tumor volume compared to inhibition of EGR1 or GDF15 alone. Our study showed that the GDF15-EGR1 signaling axis may be a good target in HNC patients.

Postretrieval Relearning Strengthens Hippocampal Memories via Destabilization and Reconsolidation.

Memory reconsolidation is hypothesized to be a mechanism by which memories can be updated with new information. Such updating has previously been shown to weaken memory expression or change the nature of the memory. Here we demonstrate that retrieval-induced memory destabilization also allows that memory to be strengthened by additional learning. We show that for rodent contextual fear memories, this retrieval conditioning effect is observed only when conditioning occurs within a specific temporal window opened by retrieval. Moreover, it necessitates hippocampal protein degradation at the proteasome and engages hippocampal Zif268 protein expression, both of which are established mechanisms of memory destabilization-reconsolidation. We also demonstrate a conceptually analogous pattern of results in human visual paired-associate learning. Retrieval-relearning strengthens memory performance, again only when relearning occurs within the temporal window of memory reconsolidation. These findings link retrieval-mediated learning in humans to the reconsolidation literature, and have potential implications both for the understanding of endogenous memory gains and strategies to boost weakly learned memories.SIGNIFICANCE STATEMENT Memory reconsolidation allows existing memories to be updated with new information. Previous research has demonstrated that reconsolidation can be manipulated pharmacologically and behaviorally to impair problematic memories. In this article, we show that reconsolidation can also be exploited to strengthen memory. This is shown both in rats, in a fear memory setting, and in a human declarative memory setting. For both, the behavioral conditions necessary to observe the memory strengthening match those that are required to trigger memory reconsolidation. There are several behavioral approaches that have previously been shown convincingly to strengthen memory. The present demonstration that reconsolidation can underpin long-lasting memory improvements may both provide an underlying mechanism for such approaches and provide new strategies to boost memories.

Regulation of pro-opiomelanocortin (POMC) gene transcription by interleukin-31 via early growth response 1 (EGR-1) in HaCaT keratinocytes.

Pro-opiomelanocortin (POMC) is a large precursor protein of and ß-endorphin. POMC expressed in keratinocytes regulates various pathophysiological responses, such as pruritus in atopic dermatitis. Interleukin (IL)-31 is a T helper 2 (Th2)-derived cytokine that functions as a pruritogen, stimulating the sensory neurons in the skin. However, the regulatory mechanism underlying IL-31-induced POMC expression in keratinocytes remains largely unknown. Herein, using a 5'-serial deletion and site-specific mutation constructs of the regulatory region of POMC, we demonstrated that a putative EGR1-binding sequence (EBS) motif in POMC is required for its upregulation by IL-31 in HaCaT keratinocytes. Notably, EGR-1 directly interacted with the EBS motif in POMC. The ectopic expression of EGR-1 stimulated the POMC promoter activity, whereas the knockdown of EGR-1 expression by RNA interference reduced IL-31-induced POMC expression. Furthermore, we observed that three major mitogen-activated protein kinases, ERK, JNK, and p38 kinase, mediated IL-31-induced EGR-1 expression. In summary, our results suggest that EGR-1 trans-activates POMC in response to IL-31 stimulation in HaCaT keratinocytes.

Estrogen induces EGR1 to fine-tune its actions on uterine epithelium by controlling PR signaling for successful embryo implantation.

The harmonized actions of ovarian E2 and progesterone (P4) regulate the proliferation and differentiation of uterine cells in a spatiotemporal manner. Imbalances between these hormones often lead to infertility and gynecologic diseases. Whereas numerous factors that are involved in P4 signaling have been identified, few local factors that mediate E2 actions in the uterus have been revealed. Here, we demonstrate that estrogen induces the transcription factor, early growth response 1 ( Egr1), to fine-tune its actions in uterine epithelial cells (ECs) that are responsible for uterine receptivity for embryo implantation. In the presence of exogenous gonadotrophins, ovulation, fertilization, and embryonic development normally occur in Egr1-/- mice, but these animals experience the complete failure of embryo implantation with reduced artificial decidualization. Although serum levels of E2 and P4 were comparable between Egr1+/+ and Egr1-/- mice on d 4 of pregnancy, aberrantly reduced levels of progesterone receptor in Egr1-/- uterine ECs caused enhanced E2 activity and impaired P4 response. Ultrastructural analyses revealed that Egr1-/- ECs are not fully able to provide proper uterine receptivity. Uterine mRNA landscapes in Egr1-/- mice revealed that EGR1 controls the expression of a subset of E2-regulated genes. In addition, P4 signaling was unable to modulate estrogen actions, including those that are involved in cell-cycle progression, in ECs that were deficient in EGR1. Furthermore, primary coculture of Egr1-/- ECs with Egr1+/+ stromal cells, and vice versa, supported the notion that Egr1 is required to modulate E2 actions on ECs to prepare the uterine environment for embryo implantation. In contrast to its role in ECs, loss of Egr1 in stroma significantly reduced stromal cell proliferation. Collectively, our results demonstrate that E2 induces EGR1 to streamline its actions for the preparation of uterine receptivity for embryo implantation in mice.-Kim, H.-R., Kim, Y. S., Yoon, J. A., Yang, S. C., Park, M., Seol, D.-W., Lyu, S. W., Jun, J. H., Lim, H. J., Lee, D. R., Song, H. Estrogen induces EGR1 to fine-tune its actions on uterine epithelium by controlling PR signaling for successful embryo implantation.

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.

Furanodienone overcomes temozolomide resistance in glioblastoma through the downregulation of CSPG4-Akt-ERK signalling by inhibiting EGR1-dependent transcription.

Glioblastoma multiforme (GBM) is a highly aggressive type of brain tumour. Patients with GBM respond poorly to chemotherapy and have poor survival outcomes. Neuron-glial antigen 2 (NG2), also known as chondroitin sulphate proteoglycan 4 (CSPG4), has been shown to contribute to critical processes, such as cell survival, proliferation, and chemotherapy resistance, during glioma progression. In this study, we found that furanodienone (FUR), a diene-type sesquiterpene isolated from the rhizomes of Rhizoma curcumae, exhibited a potential cytotoxic effect on temozolomide (TMZ)-resistant GBM cells in vitro by inhibiting CSPG4 and related signalling pathways. Studies investigating the mechanism demonstrated that FUR suppressed CSPG4-Akt-ERK signalling, inflammatory responses, and cytokine levels but activated caspase-dependent pathways and mitochondrial dysfunction. Furthermore, an immunofluorescence assay and a dual-luciferase reporter assay revealed that inhibition of EGR1-mediated transcription might have contributed to the FUR-dependent blockade of CSPG4 signalling and glioma cell survival. These results established a link between FUR-induced CSPG4 inhibition and the suppression of EGR1-dependent transcription. Attenuation of ERK1/2 and cytokine signalling might have generated the EGR1-dependent negative feedback loop of the CSPG4 pathway during FUR-induced apoptosis. These findings suggested that FUR could be a therapeutic candidate for the treatment of malignant glioma via targeting CSPG4 signalling.

The differential expression of the blood group P1 -A4GALT and P2 -A4GALT alleles is stimulated by the transcription factor early growth response 1.

BACKGROUND: The P1 /P2 phenotypic polymorphism is one of the earliest blood groups discovered in humans. These blood groups have been connected to different levels of expression of the A4GALT gene in P1 and P2 red blood cells; however, the detailed molecular genetic mechanism that leads to these two phenotypes has not been established. STUDY DESIGN AND METHODS: After our previous identification of an association between the single-nucleotide polymorphisms (SNPs) rs2143918 and rs5751348 in A4GALT gene and the P1 /P2 phenotype, we conduct a survey of transcription factors that might connect these SNPs with the differential expression of the P1 -A4GALT and P2 -A4GALT alleles. An in silico analysis of potential transcription factor binding motifs within the polymorphic SNPs rs2143918 and rs5751348 genomic regions was performed, and this was followed by reporter assays examining the candidate transcription factors, gene expression profiling, electrophoretic mobility shift assays, and P1 -A4GALT and P2 -A4GALT allelic expression analysis. RESULTS: The results revealed that the differential binding of transcription factor early growth response 1 to the SNP rs5751348 genomic region with the different genotypes in the A4GALT gene leads to differential activation of P1 -A4GALT and P2 -A4GALT expression. CONCLUSION: The present investigation, together with our previous study (Lai et al., Transfusion 2014;54:3222-31), have elucidated the molecular genetic details associated with the P1 /P2 blood groups.

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 ß-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.

EGR2 is critical for peripheral naïve T-cell differentiation and the T-cell response to influenza.

Early growth response 2 (EGR2) transcription factor negatively regulates T-cell activation, in contrast to the positive regulation of this process by EGR1. Here, we unexpectedly found that EGR2 promotes peripheral naïve T-cell differentiation, with delayed T-cell receptor-induced proliferation in naïve T cells from Egr2 conditional knockout (CKO) mice and decreased production of IFN-γ, IL-4, IL-9, and IL-17A in cells subjected to T-helper differentiation. Moreover, genes that promote T-cell activation, including Tbx21 and Notch1, had decreased expression in Egr2 CKO T cells and are direct EGR2 target genes. Following influenza infection, Egr2 CKO mice had delayed viral clearance, more weight loss, and more severe pathological changes in the lung than did WT and Egr1 KO mice, with decreased production of effector cytokines, increased infiltration of antigen-specific memory-precursor CD8(+) T cells, and lower numbers of lung-resident memory CD8(+) T cells. Thus, unexpectedly, EGR2 can function as a positive regulator that is essential for naïve T-cell differentiation and in vivo T-cell responses to a viral infection.

Andrographolide ameliorates diabetic retinopathy by inhibiting retinal angiogenesis and inflammation.

BACKGROUND: Andrographolide (Andro) is the main compound distributed in medicinal herb Andrographis paniculata. This study aims to observe the amelioration of Andro on streptozotocin (STZ)-induced diabetic retinopathy (DR) in mice. METHODS: STZ-induced non-proliferative DR (NPDR) for 2 months and proliferative DR (PDR) for 5 month in C57BL/6 mice were used in this study, respectively. Retinal vessels were observed by immunofluorescence staining for cluster of differentiation 31 (CD31). Evans blue permeation assay was used to detect the breakdown of blood-retinal barrier (BRB). Real-time PCR and immune-blot were used to detect mRNA and protein expression. Enzyme-linked immunosorbent assay (ELISA) was used to detect serum tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-1ß. RESULTS: Retinal immunofluorescence staining with CD31 showed that Andro reduced the increased retinal vessels in STZ-induced PDR mice. Evans blue permeation results demonstrated that Andro attenuated the breakdown of BRB in STZ-induced NPDR mice. In STZ-induced PDR mice, Andro decreased the increased vascular endothelial growth factor (VEGF) in serum and vitreous cavity, and reduced the increased retinal mRNA expression of VEGF and its receptors. In STZ-induced NPDR mice, Andro abrogated the nuclear translocation of nuclear factor κB (NF-κB) p65 and early growth response-1 (Egr-1), and reduced the increased phospho-NF-κBp65, -inhibitor of kappa B (IκB), and -IκB kinase (IKK). Andro also decreased the increased serum and retinal mRNA expression of TNF-α, IL-6, IL-1ß, serpine1, and tissue factor (TF). CONCLUSIONS: Andro ameliorates DR via attenuating retinal angiogenesis and inflammation, and VEGF, NF-κB, and Egr1 signaling pathways all play important roles in this process.

Tumor suppressor NGX6 inhibits the growth and metastasis of multiple cancers.

Nasopharyngeal carcinoma-associated gene 6 (NGX6) is a membrane protein primarily located in the nuclear membrane and cell membrane. Several groups reported that NGX6 gene was down-regulated in nasopharyngeal carcinoma (NPC), gastric cancer, lung cancer, liver cancer, and colorectal cancer and even less in the carcinomas with metastasis. Current studies have demonstrated that NGX6 possesses various biological functions, such as regulating protein expression of related genes, involving cell signal transduction pathways, negatively controlling cell cycle progression, inhibiting angiogenesis, and increasing the sensitivity of patients to anti-cancer drugs. Some factors regulating the expression level of NGX6 gene also have been studied. The methylation of promoter of NGX6 and histone H3K9 negatively regulates its expression, similar to the function of transcription factor special protein-1 (Sp1). However, the regulatory factor early growth response gene 1 (Egr-1) is provided with positive regulation function. This review will summarize the progress of those studies on NGX6 and elucidate the potential application of NGX6 for some malignant diseases.

Zif268/egr1 gene controls the selection, maturation and functional integration of adult hippocampal newborn neurons by learning.

New neurons are continuously added to the dentate gyrus of the adult mammalian brain. During the critical period of a few weeks after birth when newborn neurons progressively mature, a restricted fraction is competitively selected to survive in an experience-dependent manner, a condition for their contribution to memory processes. The mechanisms that control critical stages of experience-dependent functional incorporation of adult newborn neurons remain largely unknown. Here, we identify a unique transcriptional regulator of the functional integration of newborn neurons, the inducible immediate early gene zif268/egr1. We show that newborn neurons in zif268-KO mice undergo accelerated death during the critical period of 2-3 wk around their birth and exhibit deficient neurochemical and morphological maturation, including reduced GluR1 expression, increased NKCC1/KCC2b chloride cotransporter ratio, altered dendritic development, and marked spine growth defect. Investigating responsiveness of newborn neurons to activity-dependent expression of zif268 in learning, we demonstrate that in the absence of zif268, training in a spatial learning task during this critical period fails to recruit newborn neurons and promote their survival, leading to impaired long-term memory. This study reveals a previously unknown mechanism for the control of the selection, functional maturation, and experience-dependent recruitment of dentate gyrus newborn neurons that depends on the inducible immediate early gene zif268, processes that are critical for their contribution to hippocampal-dependent long-term memory.

A mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK)-dependent transcriptional program controls activation of the early growth response 1 (EGR1) gene during amino acid limitation.

Amino acid (AA) limitation in mammalian cells triggers a collection of signaling cascades jointly referred to as the AA response (AAR). In human HepG2 hepatocellular carcinoma, the early growth response 1 (EGR1) gene was induced by either AA deprivation or endoplasmic reticulum stress. AAR-dependent EGR1 activation was discovered to be independent of the well characterized GCN2-ATF4 pathway and instead dependent on MEK-ERK signaling, one of the MAPK pathways. ChIP showed that constitutively bound ELK1 at the EGR1 proximal promoter region was phosphorylated after AAR activation. Increased p-ELK1 binding was associated with increased de novo recruitment of RNA polymerase II to the EGR1 promoter. EGR1 transcription was not induced in HEK293T cells lacking endogenous MEK activity, but overexpression of exogenous constitutively active MEK in HEK293T cells resulted in increased basal and AAR-induced EGR1 expression. ChIP analysis of the human vascular endothelial growth factor A (VEGF-A) gene, a known EGR1-responsive gene, revealed moderate increases in AAR-induced EGR1 binding within the proximal promoter and highly inducible binding to a site within the first intron. Collectively, these data document a novel AA-activated MEK-ERK-ELK1 signaling mechanism.

Egr-1 participates in abnormally high gdnf gene transcription mediated by histone hyperacetylation in glioma cells.

Abnormally high transcription of the glial cell-line derived neurotrophic factor (gdnf) gene in glioma cells is related to the hyperacetylation of histone H3 lysine 9 (H3K9) in its promoter region II, but the mechanism remains unclear. There are three consecutive putative binding sites for the transcription factor early growth response protein 1(Egr-1) in promoter region II of the gdnf gene, and Egr-1 participates in gdnf gene transcription activation. Here we show that the acetylation level of H3K9 at Egr-1 binding sites in gdnf gene promoter region II in rat C6 astroglioma cells was significantly higher than that in normal astrocytes, and the binding capacity was also significantly higher. In C6 astroglioma cells, gdnf gene transcription significantly decreased after Egr-1 knock-down. In addition, the deletion or mutation of the Egr-1 binding site also significantly down-regulated the activity of promoter region II of this gene in vitro. When curcumin decreased the acetylation level of H3K9 at the Egr-1 binding site, the binding of Egr-1 to promoter region II and GDNF mRNA levels significantly decreased. In contrast, trichostatin A treatment significantly increased H3K9 acetylation at the Egr-1 binding site, which significantly increased both the binding of Egr-1 with promoter region II and GDNF mRNA levels. In this context, knocking down Egr-1 significantly reduced the elevation in gdnf gene transcription. Collectively, our results demonstrate that the hyperacetylation of H3K9 at Egr-1 binding sites in promoter region II of the gdnf gene can up-regulate the binding of Egr-1 to increase gdnf gene transcription in glioma cells.

E2F1 is a novel fibrogenic gene that regulates cholestatic liver fibrosis through the Egr-1/SHP/EID1 network.

UNLABELLED: E2F transcription factor 1 (E2F1) is an important regulator of metabolic diseases; however, its role in liver function remains elusive. This study unraveled a regulatory cascade involving E2F1, early growth response-1 (Egr-1), nuclear receptor small heterodimer partner (SHP, NR0B2), and EIA-like inhibitor of differentiation 1 (EID1) in cholestatic liver fibrosis. Liver E2F1 messenger RNA (mRNA) and protein expression was strongly up-regulated in human nonalcoholic steatohepatitis (NASH) and alcohol cirrhosis; the latter was inversely correlated with diminished SHP expression. E2F1 was also highly induced by 3,5-diethoxycarbonyl-1, 4-dihydrocollidine (DDC) feeding and bile-duct ligation (BDL) in mice. E2F1-/- mice exhibited reduced biliary fibrosis by DDC as determined by Masson Trichrome and Picro Sirius red staining, and decreased serum bile acid (BA), BA pool size, and fecal BA excretion. In addition, cholestatic liver fibrosis induced by BDL, as determined by immunohistochemistry analysis of a1 collagen expression, was increased in SHP-/- mice but attenuated in hepatocyte SHP-overexpressed transgenic (STG) mice. Egr-1 exhibited marked induction in livers of SHP-/- mice compared to the wild-type mice in both sham and BDL groups, and reduction in STG livers. Egr-1 promoter was activated by E2F1, and the activation was abrogated by expression of SHP and its co-repressor EID1 in hepatoma cells Huh7, Hepa1, and stellate cells LX2. Chromatin immunoprecipitation assays further confirmed the association of E2F1, SHP, and EID1 proteins with the Egr-1 promoter, and their direct protein interactions were determined by glutathione S-transferase pull-down assays. Interestingly, E2F1 activated Egr-1 expression in a biphasic fashion as described in both human and mouse hepatocytes. CONCLUSION: E2F1 is a fibrogenic gene and could serve as a potential new diagnostic marker for nonalcoholic and alcoholic liver fibrosis/cirrhosis.

Ribonucleotide reductase M2B inhibits cell migration and spreading by early growth response protein 1-mediated phosphatase and tensin homolog/Akt1 pathway in hepatocellular carcinoma.

UNLABELLED: Ribonucleotide reductase (RR)M2B is an enzyme belonging to the ribonucleotide reductase enzyme family, which is essential for DNA synthesis and repair. RRM2B plays an important role in tumor progression and metastasis; however, little is known about the expression and underlying molecular mechanisms of RRM2B in hepatocellular carcinoma (HCC). In the present study, we report that down-regulation of RRM2B in HCC is negatively associated with intrahepatic metastasis, regardless of p53 status. Moreover, the ectopic overexpression of RRM2B decreased HCC cell migration and invasion in vitro, whereas silencing RRM2B expression resulted in increased migration and invasion in vitro and intrahepatic and lung metastasis in vivo. Additionally, knockdown of RRM2B by short hairpin RNA (shRNA) in HCC cells was associated with epithelial-mesenchymal transition (EMT), including the down-regulation of E-cadherin, and the concomitant up-regulation of N-cadherin and slug. A further experiment showed that RRM2B inhibited cell migration and spreading through regulation of the early growth response protein 1 (Egr-1)/phosphatase and tensin homolog (PTEN)/Akt1 pathway. Consistently, we also detected a significant correlation between RRM2B and E-cadherin protein expression in HCC tissues. Furthermore, Egr-1 also directly bound to the RRM2B promoter and repressed RRM2B transcription, thereby establishing a negative regulatory feedback loop. CONCLUSION: These findings indicate that RRM2B suppresses cell migration and spreading by way of modulation of the Egr-1/PTEN/Akt1 pathway.

Chronic stress enhanced fear memories are associated with increased amygdala zif268 mRNA expression and are resistant to reconsolidation.

The chronically stressed brain may present a vulnerability to develop maladaptive fear-related behaviors in response to a traumatic event. In rodents, chronic stress leads to amygdala hyperresponsivity and dendritic hypertrophy and produces a post traumatic stress disorder (PTSD)-like phenotype that includes exaggerated fear learning following Pavlovian fear conditioning and resistance to extinction. It is unknown whether chronic stress-induced enhanced fear memories are vulnerable to disruption via reconsolidation blockade, as a novel therapeutic approach for attenuating exaggerated fear memories. We used a chronic stress procedure in a rat model (wire mesh restraint for 6h/d/21d) to create a vulnerable brain that leads to a PTSD-like phenotype. We then examined freezing behavior during acquisition, reactivation and after post-reactivation rapamycin administration (i.p., 40mg/kg) in a Pavlovian fear conditioning paradigm to determine its effects on reconsolidation as well as the subsequent functional activation of limbic structures using zif268 mRNA. Chronic stress increased amygdala zif268 mRNA during fear memory retrieval at reactivation. Moreover, these enhanced fear memories were unaffected by post reactivation rapamycin to disrupt long-term fear memory. Also, post-reactivation long term memory processing was also associated with increased amygdala (LA and BA), and decreased hippocampal CA1 zif268 mRNA expression. These results suggest potential challenges for reconsolidation blockade as an effective approach in treating exaggerated fear memories, as in PTSD. Our findings also support chronic stress manipulations combined with fear conditioning as a useful preclinical approach to study a PTSD-like phenotype.

Calcineurin-Rcan1 interaction contributes to stem cell factor-mediated mast cell activation.

The receptor for stem cell factor (SCF) is expressed on mast cells and hematopoietic progenitors. SCF-induced signaling pathways remain incompletely defined. In this study, we identified calcineurin and regulator of calcineurin 1 (Rcan1) as novel components in SCF signaling. Calcineurin activity was induced in SCF-stimulated primary mouse and human mast cells. NFAT was activated by SCF in bone marrow-derived mast cells (BMMCs) and mouse bone marrow cells, which contain hematopoietic progenitors. SCF-mediated activation also induced expression of Rcan1 in BMMCs. Rcan1-deficient BMMCs showed increased calcineurin activity and enhanced transcriptional activity of NF-κB and NFAT, resulting in increased IL-6 and TNF production following SCF stimulation. These results suggest that Rcan1 suppresses SCF-induced activation of calcineurin and NF-κB. We further demonstrated that SCF-induced Rcan1 expression is dependent on the transcription factor early growth response 1 (Egr1). Interestingly, SCF-induced Egr1 was also suppressed by Rcan1, suggesting a negative regulatory loop between Egr1 and Rcan1. Together, our findings revealed that calcineurin contributes to SCF-induced signaling, leading to NFAT activation, which, together with NF-κB and Egr1, is suppressed by Rcan1. Considering the wide range of biological functions of SCF, these novel regulatory mechanisms in SCF signaling may have broad implications.