Importin Alpha Research Articles

High-Throughput MicroRNA Profiles of Permissive Madin-Darby Canine Kidney Cell Line Infected with Influenza B Viruses.

Victoria and Yamagata lineages of influenza B viruses are globally circulating in seasonal epidemics. Madin–Darby canine kidney (MDCK) cells are permissive for viral isolation and vaccine manufacture. Nevertheless, the interplay between influenza B viruses and host microRNAs has not been investigated in this cell line. Therefore, the present study aims at high-throughput analysis of canine microRNA profile upon infection of influenza B viruses. Briefly, MDCK cells were infected with Victoria or Yamagata lineage at MOI of 0.01. After being harvested at 6, 12 and 24 h post infection, microRNAs were subjected to high-throughput sequencing based on MiSeq platform (Illumina). The results demonstrated that five microRNAs including cfa-miR-197, cfa-miR-215, cfa-miR361, cfa-miR-1841, and cfa-miR-1842 were overexpressed in both Victoria and Yamagata lineage infections. Interestingly, computational prediction showed that karyopherin alpha 6 (KPNA6) was targeted by cfa-miR-197 and cfa-miR-215. Moreover, the binding sites of both microRNAs were assessed by 3′-UTR reporter assay. The results showed that only cfa-miR-197 could bind to the target sites of KPNA6, leading to suppressing luciferase activity. Additionally, silencing of KPNA6 was confirmed by overexpression of cfa-miR-197. This study provides canine microRNA responses to seasonal influenza B viruses, suggesting that virus-mediated microRNAs might play crucial roles in host gene regulation.

CDK5-dependent phosphorylation and nuclear translocation of TRIM59 promotes macroH2A1 ubiquitination and tumorigenicity

Despite the development of adjuvant therapies, glioblastoma (GBM) patients remain incurable, thus justifying the urgent need of new therapies. CDK5 plays a critical role in GBM and is a potential target for GBM. However, the mechanism by which CDK5 promotes GBM tumorigenicity remains largely unknown. Here, we identify TRIM59 as a substrate of CDK5. EGFR-activated CDK5 directly binds to and phosphorylates TRIM59, a ubiquitin ligase at serine 308, which recruits PIN1 for cis–trans isomerization of TRIM59, leading to TRIM59 binding to importin α5 and nuclear translocation. Nuclear TRIM59 induces ubiquitination and degradation of the tumor suppressive histone variant macroH2A1, leading to enhanced STAT3 signaling activation and tumorigenicity. These findings are confirmed by inhibition of CDK5-activated TRIM59 activity that results in suppression of intracranial tumor growth. Correlative expressions of the components of this pathway are clinically prognostic. Our findings suggest targeting CDK5/TRIM59 signaling axis as a putative strategy for treating GBM.

Protection from Endotoxin Shock by Selective Targeting of Proinflammatory Signaling to the Nucleus Mediated by Importin Alpha 5.

Endotoxin shock is induced by LPS, one of the most potent virulence factors of the Gram-negative bacteria that cause sepsis. It remains unknown if either proinflammatory stress-responsive transcription factors (SRTFs), ferried to nucleus by importin α5, or lipid-regulating sterol regulatory element binding proteins (SREBPs), transported to the nucleus by importin β1, mediate endotoxin shock. A novel cell-penetrating peptide targeting importin α5 while sparing importin β1 protected 80% of animals from death in response to a high dose of LPS. This peptide suppresses inflammatory mediators, liver glycogen depletion, endothelial injury, neutrophil trafficking, and apoptosis caused by LPS. In d-galactosamine-pretreated mice challenged by 700-times lower dose of LPS, rapid death through massive apoptosis and hemorrhagic necrosis of the liver was also averted by the importin α5–selective peptide. Thus, using a new tool for selective suppression of nuclear transport, we demonstrate that SRTFs, rather than SREBPs, mediate endotoxin shock.

Divalproex sodium regulates ataxin-3 translocation likely by an importin α1-dependent pathway.

Nuclear localization of ataxin-3 plays a fundamental role in seeding aggregation and the pathology of spinocerebellar ataxia type 3 (SCA3). However, very few compounds that are able to modulate the nuclear transport of ataxin-3 have been identified. In our previous study, we found that divalproex sodium (DVS) reduced heat shock-induced nuclear localization of ataxin-3. However, the mechanism of DVS in the translocation of ataxin-3 still remains unknown. There is accumulating evidence that importins are regulated by acetylation, and histone deacetylase inhibitors can interrupt this process. With this in mind, we used cells coexpressing ataxin-3 and importin α1 (encoded by KNPA2) to probe whether ataxin-3 is the shuttling cargo of importins and whether DVS plays a role in the nuclear transport of ataxin-3 through the transport protein pathway. Here, we reported that importin α1 enhanced nuclear amount of ataxin-3 and increased the aggregate formation and that DVS restored it to the normal level. Importantly, ataxin-3 is shown to directly bind to importin α1. Moreover, DVS modulated the function of importin α1 likely by altering its localization. We believe that this study provides a proof of principle for addressing the mechanism of DVS and furthers our understanding of the role of importins in the nuclear accumulation of ataxin-3 in SCA3.

SNHG8 is upregulated in esophageal squamous cell carcinoma and directly sponges microRNA-411 to increase oncogenicity by upregulating KPNA2.

BackgroundThe long noncoding RNA, small nucleolar RNA host gene 8 (SNHG8), is upregulated in multiple human cancer types. However, whether SNHG8 is aberrantly expressed in esophageal squamous cell carcinoma (ESCC) and its biological functions have yet to be elucidated. Thus, we aimed to determine the expression status of SNHG8 in ESCC, explore the effects of SNHG8 on the oncogenicity of ESCC, and investigate the potential underlying mechanisms.MethodsSNHG8 expression in ESCC tissues and cell lines was determined via reverse-transcription quantitative polymerase chain reaction. The actions of SNHG8 on the malignant characteristics of ESCC were explored using CCK-8 assay, flow-cytometric analysis, Transwell migration and invasion assays, and tumor xenografts in nude mice.ResultsSNHG8 expression was significantly higher in ESCC tissues and cell lines. High SNHG8 expression was revealed to closely correlate with primary tumor invasion depth, lymph node metastases, TNM stage, and worse overall survival among patients with ESCC. Functional investigation showed that ablation of SNHG8 notably restricted ESCC cell proliferation, migration, and invasion while inducing apoptosis in vitro and hindered tumor growth in vivo. In the meantime, SNHG8 acted as a molecular sponge of microRNA-411 (miR-411) in ESCC. Furthermore, miR-411 exerted a tumor-suppressive effect on ESCC cells, and karyopherin alpha 2 (KPNA2) turned out to be a direct target gene of miR-411. Restoring KPNA2 expression neutralized the inhibitory effects of miR-411 overexpression on the malignant behaviors of ESCC cells. Moreover, silencing of miR-411 abrogated the influence of SNHG8 downregulation in ESCC cells.ConclusionSNHG8 may play oncogenic roles in the malignancy of ESCC by sponging miR-411 to increase KPNA2 expression. The SNHG8–miR-411–KPNA2 pathway may be a novel target for the treatment of patients with ESCC and offer potential biomarkers for the diagnosis and prognosis of ESCC.

Beta-elemene inhibits breast cancer metastasis through blocking pyruvate kinase M2 dimerization and nuclear translocation.

Pyruvate kinase M2 (PKM2), playing a central role in regulating aerobic glycolysis, was considered as a promising target for cancer therapy. However, its role in cancer metastasis is rarely known. Here, we found a tight relationship between PKM2 and breast cancer metastasis, demonstrated by the findings that beta‐elemene (β‐elemene), an approved drug for complementary cancer therapy, exerted distinct anti‐metastatic activity dependent on PKM2. The results indicated that β‐elemene inhibited breast cancer cell migration, invasion in vitro as well as metastases in vivo. β‐Elemene further inhibited the process of aerobic glycolysis and decreased the utilization of glucose and the production of pyruvate and lactate through suppressing pyruvate kinase activity by modulating the transformation of dimeric and tetrameric forms of PKM2. Further analysis revealed that β‐elemene suppressed aerobic glycolysis by blocking PKM2 nuclear translocation and the expression of EGFR, GLUT1 and LDHA by influencing the expression of importin α5. Furthermore, the effect of β‐elemene on migration, invasion, PKM2 transformation, and nuclear translocation could be reversed in part by fructose‐1,6‐bisphosphate (FBP) and L‐cysteine. Taken together, tetrameric transformation and nuclear translocation of PKM2 are essential for cancer metastasis, and β‐elemene inhibited breast cancer metastasis via blocking aerobic glycolysis mediated by dimeric PKM2 transformation and nuclear translocation, being a promising anti‐metastatic agent from natural compounds.

A glycine spacer improved peptidyl-nuclear-localized efficiency for fluorescent imaging nuclear H2O2

H2O2 generated aberrantly can attack DNA in nuclei and result in oxidative DNA damage, which has been associated with many serious diseases. Therefore, it is crucial to monitor nuclear H2O2 levels. However, the vast majority of fluorescence sensors can not localize in the nucleus. In the present study, we empolyed glycine (the simplest amino acids) as a spacer to lengthen distance between N50 and NP1. Cu2+-free click reaction (through inserting dibenzocyclooctyne-maleimide) was used to connect the two parts in situ. Three spacer sequences were tested: C, CGGGGG, and CGGGGGGGGGG, and three new NLS peptides (connection spacer sequence to N50) were generated: pep5, pep6 and pep7. The fluorescence imaging results showed the nucleus-targeting efficancy of the three peptides, especially pep6, were imporved. The calculated binding energies by molecular docking simulations with importin α5 (a member of importin) were pep6 < pep5 < pep7, consistent with the fluorescence imaging results. Importantly, the probe system consisting of pep6 and NP1 was used for imaging the increase of nuclear H2O2 level upon stimulation.

Identification of the molecular determinants for nuclear import of PRV EP0.

Pseudorabies virus (PRV) early protein EP0 is a homologue of the herpes simplex virus 1 (HSV-1) immediate-early protein ICP0, which is a multifunctional protein and important for HSV-1 infection. However, the definite function of EP0 during PRV infection is not clear. In this study, to determine if EP0 might localize to the nucleus, as it is shown for its homologue in HSV-1, the subcellular localization pattern and molecular determinants for the nuclear import of EP0 were investigated. EP0 was demonstrated to predominantly target the nucleus in both PRV infected- and plasmid-transfected cells. Furthermore, the nuclear import of EP0 was shown to be dependent on the Ran-, importin α1-, α3-, α7-, β1- and transportin-1-mediated multiple pathways. Taken together, these data will open up new horizons for portraying the biological roles of EP0 in the course of PRV lytic cycle.

Analysis of the Antiproliferative Effect of Ankaferd Hemostat on Caco-2 Colon Cancer Cells via LC/MS Shotgun Proteomics Approach.

Ankaferd hemostat (ABS), a traditional herbal extract, is a hemostatic agent used for wound healing and bleeding treatment. A standardized form of plants contains many biomolecules. In recent years, previous studies have demonstrated the antineoplastic effect of ABS. In the present work, we focused on the mechanism of its antineoplastic effect over Caco-2 colon cancer cells. The LC/MS-based proteomics method was used to understand the effect of ABS at the protein level. The results were evaluated with gene ontology, protein interaction, and pathway analysis. As shown by our results, ABS altered glucose, fatty acids, and protein metabolism. Moreover, ABS affects the cell cycle machinery. Moreover, we found that ABS induced critical cancer target and suppressor proteins such as carboxyl-terminal hydrolase 1, 60S ribosomal protein L5, Tumor protein D52-like2, karyopherin alpha 2, and protein deglycase DJ-1. In conclusion, the proteomics results indicated that ABS affects various cancer targets and suppressor proteins. Moreover ABS has systematical effect on cell metabolism and cell cycle in Caco-2 cells, suggesting that it could be used as an antineoplastic agent.

MicroRNA-139 inhibits hepatocellular carcinoma cell growth through down-regulating karyopherin alpha 2

BackgroundMicroRNA-139-5p (miR-139) has been shown to play important roles in hepatocellular carcinoma (HCC) development. However, the exact mechanism of miR-139 in HCC remains largely unknown.MethodsWe investigated the function in human cell lines and patient tissue samples by experimental techniques in molecular biology including Co-IP assay, cell viability assay, quantitative real-time-PCR, et al. In addition, datasets were used to verify the results by database analysis. Statistical analysis was performed by using the GraphPad Prism 6 (GraphPad Software Inc., USA). A P value < 0.05 was defined as statistically significant.ResultsIn this study, we found that miR-139 was significantly down-regulated in HCC. MiR-139 level was negatively associated with the stage of HCC, and HCC patients with higher miR-139 level had longer overall survival (OS) than these having lower miR-139 expression. Overexpression of miR-139 led to reduced cell viability, elevated apoptosis, and decreased colony forming, migratory and invasive capacities in HCC cells, while down-regulation of miR-139 led to opposite phenotypes. MiR-139 also inhibited HCC growth in a xenograft mouse model. We identified karyopherin alpha 2 (KPNA2) as a direct target of miR-139. KPNA2 is up-regulated in HCC and higher KPNA2 level is associated with poor patient prognosis. Silencing of KPNA2 expression led to similar phenotypic changes as miR-139 overexpression. Restoration of KPNA2 attenuated the suppressive effects of miR-139 overexpression on cell viability, apoptosis, colony formation, migration and invasion. In addition, miR-139 overexpression and KPNA2 depletion led to decreased nucleus level of POU class 5 homeobox 1 (POU5F1) and c-myc, two well-known pro-oncogenes.ConclusionIn together, these data revealed the essential roles of the miR-139/KPNA2 axis in HCC.

Karyopherin Alpha 2-Expressing Pancreatic Duct Glands and Intra-Islet Ducts in Aged Diabetic C414A-Mutant-CRY1 Transgenic Mice.

Our earlier studies demonstrated that cysteine414- (zinc-binding site of mCRY1-) alanine mutant mCRY1 transgenic mice (Tg mice) exhibit diabetes characterized by the reduction of β-cell proliferation and by β-cell dysfunction, presumably caused by senescence-associated secretory phenotype- (SASP-) like characters of islets. Earlier studies also showed that atypical duct-like structures in the pancreas developed age-dependently in Tg mice. Numerous reports have described that karyopherin alpha 2 (KPNA2) is highly expressed in cancers of different kinds. However, details of the expression of KPNA2 in pancreatic ductal atypia and in normal pancreatic tissues remain unclear. To assess the feature of the expression of KPNA2 in the development of the ductal atypia and islet architectures, we scrutinized the pancreas of Tg mice histopathologically. Results showed that considerable expression of KPNA2 was observed in pancreatic β-cells, suggesting its importance in maintaining the functions of β-cells. In mature stages, the level of KPNA2 expression was lower in islets of Tg mice than in wild-type controls. At 4 weeks, the expression levels of KPNA2 in islets of Tg mice were the same as those in wild-type controls. These results suggest that the reduction of KPNA2 might contribute to β-cell dysfunction in mature Tg mice. Additionally, the formation of mucin-producing intra-islet ducts, islet fibrosis, and massive T cell recruitment to the islet occurred in aged Tg mice. In exocrine areas, primary pancreatic intraepithelial neoplasias (PanINs) with mucinous pancreatic duct glands (PDGs) emerged in aged Tg mice. High expression of KPNA2 was observed in the ductal atypia. By contrast, KPNA2 expression in normal ducts was quite low. Thus, upregulation of KPNA2 seemed to be correlated with progression of the degree of atypia in pancreatic ductal cells. The SASP-like microenvironment inside islets might play stimulatory roles in the formation of ductal metaplasia inside islets and in islet fibrosis in Tg mice.

The Shigella type three secretion system effector OspF invades host nucleus by binding host importin α1.

Shigella type III effector OspF, a nuclear translocation protein, has been showed to have an essential role in Shigella flexneri infection and virulence; OspF increases inflammation and induces bacterial invasion by down-regulating MAPK kinases pathway. Nevertheless, the exact mechanisms underlying its nuclear translocation and signaling remain unclear. In the following study, we constructed series of OspF truncated mutants to identify the OspF nuclear localization signals which mapped to the amino acids 209-239. Moreover, we found that this sequence was essential to bind the canonical nuclear import adaptor importin α1. Additionally, the 209-239 residues deletion mutant could not bind the importin α1. Consistent with this observation, our results revealed that OspF can inhibit the MAPK signals in the nucleus. Moreover, OspF translocate to the nucleus through Ran-GTPase and importin α1-dependent manner. These results can provide new avenues for depicting the biological functions of OspF during Shigella infection.

Dual cellular localization of the Leishmania amazonensis Rbp38 (LaRbp38) explains its affinity for telomeric and mitochondrial DNA

Rbp38 is a protein exclusively found in trypanosomatid parasites, including Leishmania amazonensis, the etiologic agent of tegumentar leishmaniasis in the Americas. The protein was first described as a Leishmania tarentolae mitochondrial RNA binding protein. Later, it was shown that the trypanosomes Rbp38 orthologues were exclusively found in the mitochondria and involved in the stabilization and replication of kinetoplast DNA (kDNA). In contrast, L. amazonensis Rbp38 (LaRbp38), co-purifies with telomerase activity and interacts not only with kDNA but also with telomeric DNA, although shares with its counterparts high sequence identity and a putative N-terminal mitochondrial targeting signal (MTS). To understand how LaRbp38 interacts both with nuclear and kDNA, we have first investigated its subcellular localization. Using hydroxy-urea synchronized L. amazonensis promastigotes we could show that LaRbp38 shuttles from mitochondria to the nucleus at late S and G2 phases. Further, we identified a non-classical nuclear localization signal (NLS) at LaRbp38 C-terminal that binds with importin alpha, a protein involved in the nuclear transport of several proteins. Also, we obtained LaRbp38 truncated forms among which, some of them also showed an affinity for both telomeric DNA and kDNA. Analysis of these truncated forms showed that LaRbp38 DNA-binding region is located between amino acid residues 95–235. Together, our findings strongly suggest that LaRbp38 is multifunctional with dual subcellular localization.

Nuclear import of the DSCAM ‐cytoplasmic domain drives signaling capable of inhibiting synapse formation

DSCAM and DSCAML1 are immunoglobulin and cell adhesion-type receptors serving important neurodevelopmental functions including control of axon growth, branching, neurite self-avoidance, and neuronal cell death. The signal transduction mechanisms or effectors of DSCAM receptors, however, remain poorly characterized. We used a human ORFeome library to perform a high-throughput screen in mammalian cells and identified novel cytoplasmic signaling effector candidates including the Down syndrome kinase Dyrk1a, STAT3, USP21, and SH2D2A. Unexpectedly, we also found that the intracellular domains (ICDs) of DSCAM and DSCAML1 specifically and directly interact with IPO5, a nuclear import protein of the importin beta family, via a conserved nuclear localization signal. The DSCAM ICD is released by γ-secretase-dependent cleavage, and both the DSCAM and DSCAML1 ICDs efficiently translocate to the nucleus. Furthermore, RNA sequencing confirms that expression of the DSCAM as well as the DSCAML1 ICDs alone can profoundly alter the expression of genes associated with neuronal differentiation and apoptosis, as well as synapse formation and function. Gain-of-function experiments using primary cortical neurons show that increasing the levels of either the DSCAM or the DSCAML1 ICD leads to an impairment of neurite growth. Strikingly, increased expression of either full-length DSCAM or the DSCAM ICD, but not the DSCAML1 ICD, significantly decreases synapse numbers in primary hippocampal neurons. Taken together, we identified a novel membrane-to-nucleus signaling mechanism by which DSCAM receptors can alter the expression of regulators of neuronal differentiation and synapse formation and function. Considering that chromosomal duplications lead to increased DSCAM expression in trisomy 21, our findings may help uncover novel mechanisms contributing to intellectual disability in Down syndrome.

Importin β4 Mediates Nuclear Import of GRF-Interacting Factors to Control Ovule Development in Arabidopsis.

Ovule development is critical for seed development and plant reproduction. Multiple transcription factors (TFs) have been reported to mediate ovule development. However, it is not clear which intracellular components regulate these TFs during ovule development. After their synthesis, TFs are transported into the nucleus a process regulated by karyopherins commonly known as importin alpha and β. Around half of Arabidopsis (Arabidopsis thaliana) importin β-coding genes have been functionally characterized but only two with specific cargos have been identified. We report here that Arabidopsis IMPORTIN β4 (IMB4) regulates ovule development through nucleocytoplasmic transport of transcriptional coactivator growth regulating factors-interacting factors (GIFs). Mutations in IMB4 impaired ovule development by affecting integument growth. imb4 mutants were also defective in embryo sac development, leading to partial female sterility. IMB4 directly interacts with GIFs and is critical for the nucleocytoplasmic transport of GIF1. Finally, functional loss of GIFs resulted in ovule defects similar to those in imb4 mutants, whereas enhanced expression of GIF1 partially restored the fertility of imb4 The results presented here uncover a novel genetic pathway regulating ovule development and reveal the upstream regulator of GIFs.

Sumoylated α-synuclein translocates into the nucleus by karyopherin α6

α-Synuclein (α-SYN) is uniquely found in the brains of Parkinson’s disease (PD) patients and it has been the major target of PD research for many years. The translocation of α-SYN into the nucleus of neuronal cells is a well-known fact but the mode of nuclear entry and its functions thereafter are not well defined. By utilizing a model cell line and primary cultured neurons, we have investigated a plausible mechanism of α-SYN nuclear translocation and its pathophysiological roles in the nucleus of cells with neuronal phenotypes. We, first, identified that cytosolic α-SYN proteins bind to karyopherin alpha6 proteins only after sumoylation (i.e. SUMO2/3 binding) and then transits through the nuclear pore complex (NPC) as a complexed entity. We investigated that the nuclear translocation of α-SYN is critically required for its nuclear translocation and that this process is mediated by karyopherin alpha6.

Nucleocytoplasmic shuttling of the human parainfluenza virus type 2 phosphoprotein

Human parainfluenza virus type 2 phosphoprotein (P) is an essential component of viral polymerase. The P gene encodes both P and accessory V proteins by a specific gene editing mechanism. Therefore, the N-terminal 164 amino acids of P protein are common to V protein. Interestingly, while P protein is located in the cytoplasm, V protein is found mainly in the nucleus. Using deletion mutants, we show the presence of a nuclear localization signal (NLS) in the P/V common domain, and a nuclear export signal (NES) in the C-terminal P specific region. The NLS region makes a complex with importin α5 or 7. In the presence of leptomycin B, P protein is retained in the nucleus, indicating that it contains a CRM1-dependent NES. We identified the NLS (65PVKPRRKK72) and the NES (225IIELLKGLDL234) using β-galactosidase fusion proteins. Moreover, nucleocytoplasmic shuttling of P protein appears to be important for efficient viral polymerase activity.

Targeting TRIM3 deletion-induced tumor-associated lymphangiogenesis prohibits lymphatic metastasis in esophageal squamous cell carcinoma.

Tumor-associated lymphangiogenesis has attracted increasing attention because of its potential contribution to lymph node metastasis. However, the molecular mechanisms underlying lymphangiogenesis in cancer remains elusive. In the current study, we demonstrate that tripartite motif-containing 3 (TRIM3) directly interacts with and induces E3 ligase-dependent proteasomal turnover of importin α3 and α-Actinin-4 (ACTN4), which controls nuclear factor kappa B (NF-κB) activity at a well-ordered level. Heterozygous deletion-mediated TRIM3 downregulation led to NF-κB constitutive activation through disruption of the NF-κB-IκB-α negative feedback loop and enhancement of the p65 DNA-binding affinity and transcriptional activity via promoting symmetrical dimethylarginine modification of NF-κB/p65 at Arg30 and Arg35, which consequently promoted lymphatic metastasis of esophageal squamous cell carcinoma (ESCC) cells. Treatment with Tecfidera, a medication used to treat multiple sclerosis, restored the negative feedback inhibition of NF-κB by reducing the NF-κB/ACTN4 interaction and decreasing symmetrically dimethylated NF-κB levels, resulting in inhibition of ESCC lymphatic metastasis both in vitro and in vivo. Taken together, our results uncover a novel mechanism for constitutive NF-κB activation in cancer and may represent an attractive strategy to treat ESCC lymphatic metastasis.

Importin α5 Regulates Anxiety through MeCP2 and Sphingosine Kinase 1

SummaryImportins mediate transport from synapse to soma and from cytoplasm to nucleus, suggesting that perturbation of importin-dependent pathways should have significant neuronal consequences. A behavioral screen on five importin α knockout lines revealed that reduced expression of importin α5 (KPNA1) in hippocampal neurons specifically decreases anxiety in mice. Re-expression of importin α5 in ventral hippocampus of knockout animals increased anxiety behaviors to wild-type levels. Hippocampal neurons lacking importin α5 reveal changes in presynaptic plasticity and modified expression of MeCP2-regulated genes, including sphingosine kinase 1 (Sphk1). Knockout of importin α5, but not importin α3 or α4, reduces MeCP2 nuclear localization in hippocampal neurons. A Sphk1 blocker reverses anxiolysis in the importin α5 knockout mouse, while pharmacological activation of sphingosine signaling has robust anxiolytic effects in wild-type animals. Thus, importin α5 influences sphingosine-sensitive anxiety pathways by regulating MeCP2 nuclear import in hippocampal neurons.

Blocking Dimeric Pyruvate Kinase M2 Transformation and Nuclear Translocation Leading to Suppression of Aerobic Glycolysis Mediates Beta-Elemene Inhibition of Breast Cancer Metastasis

Pyruvate kinase M2 (PKM2) plays a central role in regulating aerobic glycolysis and is considered as a potential target for cancer therapy. However, its role in cancer metastasis is rarely known. Here, we found a tight relationship between PKM2 and breast cancer metastasis, which is demonstrated by the finding that beta-elemene (β-elemene), an approved drug for complementary cancer therapy, exerted distinct anti-metastatic activity dependent on PKM2. The results indicated thatβ-elemene inhibited breast cancer cell migration, invasion and angiogenesis. β-elemene also inhibited the process of glycolysis, and decreased the utilization of glucose and the production of pyruvate and lactate through suppressing pyruvate kinase activity by modulating the transformation of dimeric and tetrameric forms of PKM2, which was reversed in part by fructose-1,6-bisphosphate (FBP) or L-cysteine. Our further analysis revealed that β-elemene suppressed aerobic glycolysis through inhibiting the nuclear transportation of PKM2 and the expression of GLUT1, MCT1, MCT4 and LDHA by influencing the expression of importin α5. Additionally, β-elemene remarkably inhibited the breast cancer metastases in lung and liver and selectively influenced the glycolysis of the lung and liver metastatic foci in mouse models. Taken together, tetrameric transformation and nuclear translocation of PKM2 are essential for cancer metastasis, and β-elemene inhibits breast cancer metastasis via blocking aerobic glycolysis induced by dimeric PKM2 transformation and EGFR-importin α5 mediated PKM2 nuclear translocation, being a promising anti-metastatic agent. Funding Statement: The work was supported by National Natural Science Foundation of China (No. 81673648, 81673725, 81573859, 81603339), Natural Science Foundation of Higher School of Jiangsu Province (17KJA360003), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). Declaration of Interests: The authors declare that they have no conflict of interest. Ethics Approval Statement: All animal studies were approved by the Animal Care and Use Committee of Nanjing University of Chinese Medicine. All mouse experimental procedures were performed in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals approved by the State Council of People’s Republic of China.