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ABSTRACT: Herpes simplex virus type 1 (HSV-1) replicates in various cell types and induces early cell death, which limits viral replication in certain cell types. Axin is a scaffolding protein that regulates Wnt signaling and participates in various cellular events including cellular proliferation and cell death. The effects of axin expression on HSV-1 infection were investigated based on our initial observation that Wnt3a treatment or axin knockdown reduced HSV-1 replication. L929 cells expressed the axin protein in a doxycycline-inducible manner (L-axin) and enhanced HSV-1 replication in comparison to control cells (L-EV). HSV-1 infection induced cell death as early as 6h after infection through the necrotic pathway and required de novo protein synthesis in L929 cells. Subsequent analysis of viral protein expression suggested that axin expression led to suppression of HSV-1-induced premature cell death, resulting in increased late gene expression. In analysis of axin deletion mutants, the regulators of G-protein signaling (RGS) domain was involved in axin-mediated enhancement of viral replication and reduction in cell death. These results suggest that enhancing viral replication might be mediated by the axin RGS domain.
Journal of General Virology 03/2013; · 3.36 Impact Factor
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ABSTRACT: Wnt/β-catenin signalling plays essential roles in embryonic development as well as tissue homoeostasis in adults. Thus abnormal regulation of Wnt/β-catenin signalling is linked to a variety of human diseases, including cancer, osteoporosis and Alzheimer's disease. Owing to the importance of Wnt signalling in a wide range of biological fields, a better understanding of its precise mechanisms could provide fundamental insights for therapeutic applications. Although many studies have investigated the regulation of Wnt/β-catenin signalling, our knowledge remains insufficient due to the complexity and diversity of Wnt signalling. It is generally accepted that the identification of novel regulators and their functions is a prerequisite to fully elucidating the regulation of Wnt/β-catenin signalling. Recently, several novel modulators of Wnt signalling have been determined through multiple genetic and proteomic approaches. In the present review, we discuss the mechanistic regulation of Wnt/β-catenin signalling by focusing on the roles of these novel regulators.
Biochemical Journal 02/2013; 450(1):9-21. · 4.90 Impact Factor
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ABSTRACT: DP1, a dimerization partner protein of the transcription factor E2F, is known to inhibit Wnt/β-catenin signalling along with E2F, although the function of DP1 itself was not well characterized. Here, we present a novel dual regulatory mechanism of Wnt/β-catenin signalling by DP1 independent from E2F. DP1 negatively regulates Wnt/β-catenin signalling by inhibiting Dvl-Axin interaction and by enhancing poly-ubiquitination of β-catenin. In contrast, DP1 positively modulates the signalling upon Wnt stimulation, via increasing cytosolic β-catenin and antagonizing the kinase activity of NLK. In Xenopus embryos, DP1 exerts both positive and negative roles in Wnt/β-catenin signalling during anteroposterior neural patterning. From subcellular localization analyses, we suggest that the dual roles of DP1 in Wnt/β-catenin signalling are endowed by differential nucleocytoplasmic localizations. We propose that these dual functions of DP1 can promote and stabilize biphasic Wnt-on and Wnt-off states in response to a gradual gradient of Wnt/β-catenin signalling to determine differential cell fates.
The EMBO Journal 07/2012; 31(16):3384-97. · 9.20 Impact Factor
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ABSTRACT: INTRODUCTION: Protein arginine methyltransferases (PRMTs) add one or two monomethyl groups to the guanidino nitrogen atoms of arginine residues, resulting in epigenetic modification of histones or changes of protein-protein interactions, which in turn leads to the regulation of a variety of biological functions, including transcriptional activation/repression, signal transduction, cell differentiation, and embryonic development. As dysregulation of PRMTs has been observed in diverse types of cancers and modulation of their levels affects cancer cell growth, these enzymes are considered to be potential therapeutic targets. AREAS COVERED: In this review, we examined recent advances in our understanding of the regulatory mechanisms of PRMT activity and the biological roles of PRMTs in embryonic stem cell, Wnt/β-catenin signaling, and cancer development. EXPERT OPINION: The roles of PRMTs have been fairly well established, but further studies are required to determine how PRMTs are regulated by cellular signaling pathways in vivo. Since the usage of adult stem cells is under intense scrutiny by society, identification of the roles of PRMTs in adult stem cells is expected in the near future. Although small molecules specific to PRMTs with high potency in vitro have been identified, development of small molecules that can regulate the activity of PRMTs in vivo is urgently required for therapeutic purposes.
Expert opinion on therapeutic targets 05/2012; 16(7):651-64. · 3.72 Impact Factor
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Hanjun Kim,
Sehoon Won,
Dae Youn Hwang,
Jeong-sook Lee,
Minseong Kim,
Rocky Kim,
Wantae Kim,
Boksik Cha,
Taeyoon Kim,
Daesoo Kim,
Frank Costantini, Eek-hoon Jho
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ABSTRACT: The possibility that the degeneration of hippocampal neurons can be caused by mis-regulation of Wnt/β-catenin signaling was tested. Downregulation of Wnt signaling by the inducible expression of Axin, ICAT, and dnTcf4E causes degeneration of hippocampal neurons, while upregulation of Wnt signaling by the inducible expression of Dvl and β-catenin has a negligible effect. Treatment with ICG-001, a small molecule known to inhibit Wnt signaling, causes degeneration of hippocampal neurons, while the treatment with a JNK specific inhibitor does not show any effect. The results from LDH and TUNEL assays suggest that degeneration occurs via apoptotic processes. Inhibition of Wnt signaling reduced IGF-1 expression and the addition of IGF-1 blocked degeneration, which suggests that downregulation of IGF-1/Akt signaling is partially responsible for the degeneration. Inducible expression of Axin in the hippocampal neurons isolated from Axin2P-rtTA/pBI-EGFP-Axin double transgenic mice also causes degeneration. Consistent with the findings, these mice had more neuronal cell death in hippocampus and had differences in contextual conditioning upon the inducible expression of Axin. In summary, our data strongly support the idea that downregulation of Wnt/β-catenin signaling causes degeneration of hippocampal neurons in vivo and may be a cause of neurodegenerative diseases related to an anxiety related response.
Neurobiology of aging 12/2011; 32(12):2316.e1-15. · 5.94 Impact Factor
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ABSTRACT: Platelet aggregation plays crucial roles in the formation of hemostatic plugs and thrombosis. Although it was recently shown that canonical Wnt signaling negatively regulates platelet aggregation, the role of non-canonical Wnt signaling remains unknown. Here, we observed that Wnt5a, one of the non-canonical Wnts, positively regulated platelet aggregation. Platelet aggregation was potentiated by the addition of Wnt5a to collagen-or U46619-induced rat platelet rich plasma (PRP). Treatment with Wnt5a to U46619-stimulated PRP resulted in an increase in the level of phosphorylated Akt, whereas phosphorylation of PKCδ and JNK1 was unaffected. In addition, inhibition of PI3K blocked the potentiating effect of Wnt5a. Taken together, these results suggest that Wnt5a potentiates U46619-induced platelet aggregation via the PI3K/Akt pathway.
Molecules and Cells 08/2011; 32(4):333-6. · 2.18 Impact Factor
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ABSTRACT: Mest (mesoderm-specific transcript)/Peg1 (paternally expressed gene 1) is an imprinted gene that plays important roles in embryo development, although its biochemical role has not been determined. Ectopic expression of Mest/Peg1 inhibited Wnt-mediated reporter activity by enhancing the ubiquitination of β-catenin. The maturation and plasma membrane localization of the Wnt co-receptor LRP6 [LDLR (low-density lipoprotein receptor)-related protein 6], which are both necessary for Wnt signalling, were blocked by the expression of Mest/Peg1. Mest/Peg1 inhibited maturation of LRP6 by controlling the glycosylation of LRP6. Knockdown of Mest/Peg1, which might enhance Wnt signalling, blocked adipogenic differentiation of 3T3-L1 cells. Overall, our results suggest that Mest/Peg1 is a novel regulator of Wnt/β-catenin signalling during adipogenic differentiation.
Biochemical Journal 03/2011; 436(2):263-9. · 4.90 Impact Factor
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ABSTRACT: In embryonic stem cells (ESCs), Wnt-responsive development-related genes are silenced to maintain pluripotency and their expression is activated during differentiation. Acetylation of histones by histone acetyltransferases stimulates transcription, whereas deacetylation of histones by HDACs is correlated with transcriptional repression. Although Wnt-mediated gene transcription has been intimately linked to the acetylation or deacetylation of histones, how Wnt signaling regulates this type of histone modification is poorly understood. Here, we report that Smek, a regulatory subunit of protein phosphatase 4 (PP4) complex, plays an important role in histone deacetylation and silencing of the Wnt-responsive gene, brachyury, in ESCs. Smek mediates recruitment of PP4c and HDAC1 to the Tcf/Lef binding site of the brachyury promoter and inhibits brachyury expression in ESCs. Activation of Wnt signaling during differentiation causes disruption of the Smek/PP4c/HDAC1 complex, resulting in an increase in histones H3 and H4 acetylation at the brachyury gene locus. These results suggest that the Smek-containing PP4 complex represses transcription of Wnt-responsive development-related genes through histone deacetylation, and that this complex is essential for ESC pluripotency maintenance.
Cell Research 03/2011; 21(6):911-21. · 8.19 Impact Factor
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ABSTRACT: Impaired responsiveness of platelets to epinephrine (epi) and other catecholamines (CA) has been reported in approximately 20% of the healthy Korean and Japanese populations. In the present study, platelet aggregation induced by epi was potentiated by RO 31-8220 (RO) or Gö 6983 (Gö). Phosphorylated Akt (p-Akt) was very low in epi-stimulated PRP from CA-hypo-responders (CA-HY), whereas it was detected in those from CA-good responders (CA-GR). RO and Gö increased p-Akt, one of the major downstream effectors of phosphoinositol-3 kinase (PI3K), in epi-stimulated PRP from both groups. Wortmannin, a PI3K inhibitor, attenuated the RO or Gö-induced potentiation of p-Akt in epi-stimulated PRP, suggesting positive effects for RO and Gö on PI3K. TXA(2) formation was increased by the addition of either RO or Gö in epi-stimulated platelets. The present data also suggest that impaired Akt phosphorylation may be responsible for epinephrine hypo-responsiveness of platelets.
BMB reports 02/2011; 44(2):140-5. · 1.72 Impact Factor
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ABSTRACT: Axin is a negative regulator of Wnt/β-catenin signaling via regulating the level of β-catenin, which is a key effector molecule. Therefore, controlling the level of Axin is a critical step for the regulation of Wnt/β-catenin signaling. It has been shown that ubiquitination-mediated proteasomal degradation may play a critical role in the regulation of Axin; however, the E3 ubiquitin ligase(s), which attaches ubiquitin to a target protein in combination with an E2 ubiquitin-conjugating enzyme, for Axin has not yet been identified. Here, we show that Smurf2 is an E3 ubiquitin ligase for Axin. Transient expression of Smurf2 down-regulated the level of Axin and increased the ubiquitination of Axin. Conversely, shRNA specific to Smurf2 blocked Axin ubiquitination. Essential domains of Axin responsible for Smurf2 interaction as well as Smurf2-mediated down-regulation and ubiquitination were identified. In vitro ubiquitination assays followed by analysis using mass spectroscopy revealed that Smurf2 specifically ubiquitinylated Lys(505) of Axin and that the Axin(K505R) mutant resisted degradation. Knockdown of endogenous Smurf2 increased the level of endogenous Axin and resulted in reduced β-catenin/Tcf reporter activity. Overall, our data strongly suggest that Smurf2 is a genuine E3 ligase for Axin.
Journal of Biological Chemistry 11/2010; 285(47):36420-6. · 4.77 Impact Factor
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ABSTRACT: Wnt/β-catenin signaling plays critical roles in embryonic development and tissue homeostasis in adults by controlling the expression of target genes. We found that expression of ptpro, which encodes a protein tyrosine phosphatase receptor type O (PTPRO), was induced by Wnt/β-catenin signaling in a T cell factor/lymphoid enhancer factor dependent manner. Biochemical assays found that PTPRO interacted with Wnt via its extracellular domain. In addition, ectopic expression of this extracellular domain inhibited Wnt-mediated reporter activity. These results suggest that ptpro is a target gene of Wnt/β-catenin signaling and that PTPRO may function as a novel receptor for Wnt.
FEBS letters 09/2010; 584(18):3923-8. · 3.54 Impact Factor
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ABSTRACT: Controlled gene expression in specific cells is a valuable tool for gene therapy. We attempted to determine whether the lentivirus-mediated Tet-On inducible system could be applied to cancer gene therapy. In order to select the genes that induce cancer cell death, we compared the ability of the known pro-apoptotreic genes, Bax and tBid, and a cell cycle inhibitor, p21cip1/waf1, and determined that Bax was the most effective. For the cancer cell-specific expression of rtTA2(S)-M2, we tested the matrix metalloproteinase-2 (MMP-2) promoter and determined that it is highly expressed in cancer cell lines, including SNU475 cells. The co-transduction of two lentiviruses that contain sequences for TRE-Bax and rtTA2(S)-M2, the expression of which is controlled by the MMP-2 promoter, resulted in the specific cell death of SNU475, whereas other cells with low MMP-2 expression did not evidence significant cell death. Our data indicate that the lentivirus-mediated Tet-On system using the cancer-specific promoter is applicable for cancer gene therapy.
BMB reports 05/2009; 42(4):217-22. · 1.72 Impact Factor
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ABSTRACT: Wnt signaling is implicated in a variety of developmental and pathological processes. The molecular mechanisms governing the secretion of Wnt ligands remain to be elucidated. Wntless, an evolutionarily conserved multipass transmembrane protein, is a dedicated secretion factor of Wnt proteins that participates in Drosophila melanogaster embryogenesis. In this study, we show that Xenopus laevis Wntless (XWntless) regulates the secretion of a specific Wnt ligand, XWnt4, and that this regulation is specifically required for eye development in Xenopus. Moreover, the Retromer complex is required for XWntless recycling to regulate the XWnt4-mediated eye development. Inhibition of Retromer function by Vps35 morpholino (MO) resulted in various Wnt deficiency phenotypes, affecting mesoderm induction, gastrulation cell movements, neural induction, neural tube closure, and eye development. Overexpression of XWntless led to the rescue of Vps35 MO-mediated eye defects but not other deficiencies. These results collectively suggest that XWntless and the Retromer complex are required for the efficient secretion of XWnt4, facilitating its role in Xenopus eye development.
Molecular and cellular biology 03/2009; 29(8):2118-28. · 6.06 Impact Factor
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Jin-A Kim,
Young-Ju Kang,
Gyeongsin Park,
Myungshin Kim,
Young-Ok Park,
Hanjun Kim,
Sun-Hee Leem,
In-Sun Chu,
Jun-Seong Lee, Eek-Hoon Jho,
Il-Hoan Oh
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ABSTRACT: With contrasting observations on the effects of beta-catenin on hematopoietic stem cells (HSCs), the precise role of Wnt/beta-catenin signals on HSC regulation remains unclear. Here, we show a distinct mode of Wnt/beta-catenin signal that can regulate HSCs in a stroma-dependent manner. Stabilization of beta-catenin in the bone marrow stromal cells promoted maintenance and self-renewal of HSCs in a contact-dependent manner, whereas direct stabilization in hematopoietic cells caused loss of HSCs. Interestingly, canonical Wnt receptors and beta-catenin accumulation were predominantly enriched in the stromal rather than the hematopoietic compartment of bone marrows. Moreover, the active form of beta-catenin accumulated selectively in the trabecular endosteum in "Wnt 3a-stimulated" or "irradiation-stressed," but not in "steady-state" marrows. Notably, notch ligands were induced in Wnt/beta-catenin activated bone marrow stroma and downstream notch signal activation was seen in the HSCs in contact with the activated stroma. Taken together, Wnt/beta-catenin activated stroma and their cross-talk with HSCs may function as a physiologically regulated microenvironmental cue for HSC self-renewal in the stem cell niche.
Stem Cells 03/2009; 27(6):1318-29. · 7.78 Impact Factor
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ABSTRACT: Wnt signaling plays critical roles in cell proliferation and carcinogenesis. In addition, numerous recent studies have shown that various Wnt signaling components are involved in mitosis and chromosomal instability. However, the role of Axin, a negative regulator of Wnt signaling, in mitosis has remained unclear. Using monoclonal antibodies against Axin, we found that Axin localizes to the centrosome and along mitotic spindles. This localization was suppressed by siRNA specific for Aurora A kinase and by Aurora kinase inhibitor. Interestingly, Axin over-expression altered the subcellular distribution of Plk1 and of phosphorylated glycogen synthase kinase (GSK3beta) without producing any notable changes in cellular phenotype. In the presence of Aurora kinase inhibitor, Axin over-expression induced the formation of cleavage furrow-like structures and of prominent astral microtubules lacking midbody formation in a subset of cells. Our results suggest that Axin modulates distribution of Axin-associated proteins such as Plk1 and GSK3beta in an expression level-dependent manner and these interactions affect the mitotic process, including cytokinesis under certain conditions, such as in the presence of Aurora kinase inhibitor.
Experimental Cell Research 02/2009; 315(6):943-54. · 3.58 Impact Factor
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ABSTRACT: The F-box proteins beta-TrCP1 and 2 (beta-transducin repeat containing protein) have 2 and 3 isoforms, respectively, due to alternative splicing of exons encoding the N-terminal region. We identified an extra exon in between the previously known exons 1 and 2 of beta-TrCP1 and beta-TrCP2. Interestingly, sequence analysis suggested that many more isoforms are produced than previously identified, via the alternative splicing of all possible combination of exons II to V of beta-TrCP1 and exons II to IV of beta-TrCP2. Different mouse tissues show specific expression patterns of the isoforms, and the level of expression of the isoform that has been used in most published papers was very low. Yeast two-hybrid assays show that beta-TrCP1 isoforms containing exon III, which are the most highly expressed isoforms in most tissues, do not interact with Skp1. Indirect immunofluorescence analysis of transiently expressed beta-TrCP1 isoforms suggests that the presence of exon III causes beta-TrCP1 to localize in nuclei. Consistent with the above findings, isoforms including exon III showed a reduced ability to block ectopic embryonic axes induced via injection of Wnt8 or beta-catenin in Xenopus embryos. Overall, our data suggest that isoforms of beta-TrCPs generated by alternative splicing may have different biological roles.
Cellular signalling 10/2008; 21(1):43-51. · 4.09 Impact Factor
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ABSTRACT: Microarray analysis was carried out to identify novel downstream target genes regulated by non-canonical Wnt signaling. We found that Tmod1, known as an actin-capping protein, is up-regulated by Wnt5a, while gelsolin, known as an actin-severing protein, is down-regulated by Wnt5a. As expected from the roles of genes regulated by Wnt5a, L929 cells expressing Wnt5a show abnormal cell shape and a reduced migration rate. Cloning and analysis of the putative promoter show that two conserved sequences, one in the 5'-end of the first exon and the other in the intron next to the first exon, are necessary for the basal promoter activity.
Biochemical and Biophysical Research Communications 12/2007; 363(3):727-32. · 2.48 Impact Factor
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ABSTRACT: Interactions between the Wnt/beta-catenin and the extracellular signal-regulated kinase (ERK) pathways have been posited, but the molecular mechanisms and cooperative roles of such interaction in carcinogenesis are poorly understood. In the present study, the Raf-1, MEK, and ERK activities were concomitantly decreased in fibroblasts, which inhibit morphological transformation and proliferation by Axin induction. The inhibition of the components of the ERK pathway by Axin occurred in cells retaining wild-type beta-catenin, including primary hepatocytes, but not in cells retaining non-degradable mutant beta-catenin. Axin inhibits cellular proliferation and ERK pathway activation induced by either epidermal growth factor or Ras, indicating a role of Axin in the regulation of growth induced by ERK pathway activation. ERK pathway regulation by Axin occurs at least partly via reduction of the protein level of Ras. Both wild-type and mutant Ras proteins are subjected to regulation by Axin, which occurs in cells retaining wild-type but not mutant beta-catenin gene. The role of beta-catenin in the regulation of the Ras-ERK pathway was further confirmed by Ras reduction and subsequent inhibitions of the ERK pathway components by knock down of mutated form of beta-catenin. The Ras regulation by Axin was blocked by treatment of leupeptin, an inhibitor of the lysosomal protein degradation machinery. Overall, Axin inhibits proliferation of cells at least partly by reduction of Ras protein level via beta-catenin. This study provides evidences for the role of the Ras-ERK pathway in carcinogenesis caused by mutations of the Wnt/beta-catenin pathway components.
Journal of Biological Chemistry 06/2007; 282(19):14482-92. · 4.77 Impact Factor
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ABSTRACT: It has been shown that accumulation of free beta-catenin leads to mobility shift of adenomatous polyposis coli (APC) protein and that Axin facilitates this process. Here we show that the beta-catenin-mediated mobility shift of APC is due to phosphorylation of two domains of APC by casein kinase 1epsilon/glycogen synthase kinase 3beta and unknown kinase(s), respectively. Interestingly, our results suggest that this process does not require Axin. The phosphorylated APC showed higher affinity to beta-catenin in vivo, and fragments of APC containing the phosphorylated domains can inhibit beta-catenin/Tcf-mediated reporter activity regardless of their ability to reduce the level of beta-catenin. From our data we propose a new role of APC: accumulation of excessive cytoplasmic beta-catenin induces phosphorylation of APC and the phosphorylated APC retains beta-catenin in cytoplasm to prevent excessive beta-catenin signaling. The retained beta-catenin in cytoplasm by APC may be down-regulated by Axin 2, which is induced by beta-catenin/Tcf signaling.
Biochemical and Biophysical Research Communications 06/2007; 357(1):81-6. · 2.48 Impact Factor
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ABSTRACT: Focal adhesion kinase (FAK) mediates signal transduction in response to multiple extracellular inputs via tyrosine phosphorylation at specific residues. Although several tyrosine phosphorylation events have been linked to FAK activation and downstream signal transduction, the function of FAK phosphorylation at Tyr(407) was previously unknown. Here, we show for the first time that phosphorylation of FAK Tyr(407) increases during serum starvation, contact inhibition, and cell cycle arrest, all conditions under which activating FAK Tyr(397) phosphorylation decreases. Transfection of NIH3T3 cells with a phosphorylation-mimicking FAK 407E mutant decreased autophosphorylation at Tyr(397) and inhibited both FAK kinase activity in vitro and FAK-mediated functions such as cell adhesion, spreading, proliferation, and migration. The opposite effects were observed in cells transfected with nonphosphorylatable mutant FAK 407F. Taken together, these data suggest the novel concept that FAK Tyr(407) phosphorylation negatively regulates the enzymatic and biological activities of FAK.
Journal of Biological Chemistry 05/2007; 282(14):10398-404. · 4.77 Impact Factor
