Effects of Importin α1/KPNA1 deletion and adolescent social isolation stress on psychiatric disorder-associated behaviors in mice.

Importin α1/KPNA1 is a member of the Importin α family widely present in the mammalian brain and has been characterized as a regulator of neuronal differentiation, synaptic functionality, and anxiety-like behavior. In humans, a de novo mutation of the KPNA1 (human Importin α5) gene has been linked with schizophrenia; however, the precise roles of KPNA1 in disorder-related behaviors are still unknown. Moreover, as recent studies have highlighted the importance of gene-environment interactions in the development of psychiatric disorders, we investigated the effects of Kpna1 deletion and social isolation stress, a paradigm that models social stress factors found in human patients, on psychiatric disorder-related behaviors in mice. Through assessment in a behavioral battery, we found that Kpna1 knockout resulted in the following behavioral phenotype: (1) decreased anxiety-like behavior in an elevated plus maze test, (2) short term memory deficits in novel object recognition test (3) impaired sensorimotor gating in a prepulse inhibition test. Importantly, exposure to social isolation stress resulted in additional behavioral abnormalities where isolated Kpna1 knockout mice exhibited: (1) impaired aversive learning and/or memory in the inhibitory avoidance test, as well as (2) increased depression-like behavior in the forced swim test. Furthermore, we investigated whether mice showed alterations in plasma levels of stress-associated signal molecules (corticosterone, cytokines, hormones, receptors), and found that Kpna1 knockout significantly altered levels of corticosterone and LIX (CXCL5). Moreover, significant decreases in the level of prolactin were found in all groups except for group-housed wild type mice. Our findings demonstrate that Kpna1 deletion can trigger widespread behavioral abnormalities associated with psychiatric disorders, some of which were further exacerbated by exposure to adolescent social isolation. The use of Kpna1 knockout mice as a model for psychiatric disorders may show promise for further investigation of gene-environment interactions involved in the pathogenesis of psychiatric disorders.

Transportin-2 plays a critical role in nucleocytoplasmic shuttling of oestrogen receptor-α.

Oestrogen receptor-α (ERα) shuttles continuously between the nucleus and the cytoplasm, and functions as an oestrogen-dependent transcription factor in the nucleus and as an active mediator of signalling pathways, such as phosphatidylinositol 3-kinase (PI3K)/AKT, in the cytoplasm. However, little is known regarding the mechanism of ERα nucleocytoplasmic shuttling. In this study, we found that ERα is transported into the nucleus by importin-α/ß1. Furthermore, we found that Transportin-2 (TNPO2) is involved in 17ß-oestradiol (E2)-dependent cytoplasmic localisation of ERα. Interestingly, it was found that TNPO2 does not mediate nuclear export, but rather is involved in the cytoplasmic retention of ERα via the proline/tyrosine (PY) motifs. Moreover, we found that TNPO2 competitively binds to the basic nuclear localisation signal (NLS) of ERα with importin-α to inhibit importin-α/ß-dependent ERα nuclear import. Finally, we confirmed that TNPO2 knockdown enhances the nuclear localisation of wild-type ERα and reduces PI3K/AKT phosphorylation in the presence of E2. These results reveal that TNPO2 regulates nucleocytoplasmic shuttling and cytoplasmic retention of ERα, so that ERα has precise functions depending on the stimulation.

Two isoforms of TALDO1 generated by alternative translational initiation show differential nucleocytoplasmic distribution to regulate the global metabolic network.

Transaldolase 1 (TALDO1) is a rate-limiting enzyme involved in the pentose phosphate pathway, which is traditionally thought to occur in the cytoplasm. In this study, we found that the gene TALDO1 has two translational initiation sites, generating two isoforms that differ by the presence of the first 10 N-terminal amino acids. Notably, the long and short isoforms were differentially localised to the cell nucleus and cytoplasm, respectively. Pull-down and in vitro transport assays showed that the long isoform, unlike the short one, binds to importin α and is actively transported into the nucleus in an importin α/ß-dependent manner, demonstrating that the 10 N-terminal amino acids are essential for its nuclear localisation. Additionally, we found that these two isoforms can form homo- and/or hetero-dimers with different localisation dynamics. A metabolite analysis revealed that the subcellular localisation of TALDO1 is not crucial for its activity in the pentose phosphate pathway. However, the expression of these two isoforms differentially affected the levels of various metabolites, including components of the tricarboxylic acid cycle, nucleotides, and sugars. These results demonstrate that the nucleocytoplasmic distribution of TALDO1, modulated via alternative translational initiation and dimer formation, plays an important role in a wide range of metabolic networks.

Cell surface localization of importin α1/KPNA2 affects cancer cell proliferation by regulating FGF1 signalling.

Importin α1 is involved in nuclear import as a receptor for proteins with a classical nuclear localization signal (cNLS). Here, we report that importin α1 is localized to the cell surface in several cancer cell lines and detected in their cultured medium. We also found that exogenously added importin α1 is associated with the cell membrane via interaction with heparan sulfate. Furthermore, we revealed that the cell surface importin α1 recognizes cNLS-containing substrates. More particularly, importin α1 bound directly to FGF1 and FGF2, secreted cNLS-containing growth factors, and addition of exogenous importin α1 enhanced the activation of ERK1/2, downstream targets of FGF1 signalling, in FGF1-stimulated cancer cells. Additionally, anti-importin α1 antibody treatment suppressed the importin α1-FGF1 complex formation and ERK1/2 activation, resulting in decreased cell growth. This study provides novel evidence that functional importin α1 is located at the cell surface, where it accelerates the proliferation of cancer cells.

Retinoblastoma-binding Protein 4-regulated Classical Nuclear Transport Is Involved in Cellular Senescence.

Nucleocytoplasmic trafficking is a fundamental cellular process in eukaryotic cells. Here, we demonstrated that retinoblastoma-binding protein 4 (RBBP4) functions as a novel regulatory factor to increase the efficiency of importin α/ß-mediated nuclear import. RBBP4 accelerates the release of importin ß1 from importin α via competitive binding to the importin ß-binding domain of importin α in the presence of RanGTP. Therefore, it facilitates importin α/ß-mediated nuclear import. We showed that the importin α/ß pathway is down-regulated in replicative senescent cells, concomitant with a decrease in RBBP4 level. Knockdown of RBBP4 caused both suppression of nuclear transport and induction of cellular senescence. This is the first report to identify a factor that competes with importin ß1 to bind to importin α, and it demonstrates that the loss of this factor can trigger cellular senescence.

Functional characterization of importin α8 as a classical nuclear localization signal receptor.

Importin α8 has recently been identified as an importin α family member based on its primary structure and binding ability to importin ß1 and to several karyophilic proteins. However, there has been no experimental evidence that importin α8 actually functions in the nuclear transport of classical nuclear localization signal (cNLS)-containing cargo. Here, using an in vitro transport assay, we demonstrate that purified recombinant importin α8 can transport SV40T antigen cNLS-containing cargo into the nucleus of HeLa cells, in conjunction with importin ß1. Pull-down assays, followed by mass spectrometry analysis, identified 179 putative importin α8-binding proteins, only 62 of which overlap with those of importin α1, the closest importin α family member. Among the importin α8-binding candidates, we showed that DNA damage-binding protein 2 (DDB2) was actually transported into the nucleus via the importin α8/ß1 pathway. Furthermore, we found that the other subtypes of importin α, which were also identified as importin α8-binding candidates, indeed form heterodimers with importin α8. Notably, we found that these importin α8-containing heterodimers were more stable in the presence of cNLS-substrates than heterodimers containing importin α1. From these findings, we propose that importin α8 functions as a cNLS receptor with distinct cargo specificity, and that heterodimerization by importin α8 is a novel regulatory mode of cNLS binding, in addition to the autoinhibitory regulation by the importin ß binding domain.

Identification and characterization of a nuclear localization signal of TRIM28 that overlaps with the HP1 box.

Tripartite motif-containing 28 (TRIM28) is a transcription regulator, which forms a repressor complex containing heterochromatin protein 1 (HP1). Here, we report identification of a nuclear localization signal (NLS) within the 462-494 amino acid region of TRIM28 that overlaps with its HP1 binding site, HP1 box. GST-pulldown experiments revealed the interaction of the arginine-rich TRIM28 NLS with various importin α subtypes (α1, α2 and α4). In vitro transport assay demonstrated that nuclear localization of GFP-TRIM28 NLS is mediated by importin αs, in conjunction with importin ß1 and Ran. Further, we demonstrated that HP1 and importin αs compete for binding to TRIM28. Together, our findings suggest that importin α has an essential role in the nuclear delivery and preferential HP1 interaction of TRIM28.

Importin alpha subtypes determine differential transcription factor localization in embryonic stem cells maintenance.

We recently demonstrated that the expression of the importin α subtype is switched from α2 to α1 during neural differentiation in mouse embryonic stem cells (ESCs) and that this switching has a major impact on cell differentiation. In this study, we report a cell-fate determination mechanism in which importin α2 negatively regulates the nuclear import of certain transcription factors to maintain ESC properties. The nuclear import of Oct6 and Brn2 was inhibited via the formation of a transport-incompetent complex of the cargo bound to a nuclear localization signal binding site in importin α2. Unless this dominant-negative effect was downregulated upon ESC differentiation, inappropriate cell death was induced. We propose that although certain transcription factors are necessary for differentiation in ESCs, these factors are retained in the cytoplasm by importin α2, thereby preventing transcription factor activity in the nucleus until the cells undergo differentiation.

Differential role for transcription factor Oct4 nucleocytoplasmic dynamics in somatic cell reprogramming and self-renewal of embryonic stem cells.

Oct4 is a member of the POU family of transcription factors and plays a critical role in both maintenance of the undifferentiated state of embryonic stem (ES) cells and in the reprogramming of somatic cells to induced pluripotent stem cells. Oct4 is imported into the nucleus where it functions as a transcription factor; however, the spatiotemporal dynamic behavior of Oct4 remains largely unknown. In the present study we show that Oct4 is a nucleocytoplasmic shuttling protein. Furthermore, although Oct4 mutants with altered nuclear import/export activity were able to maintain the self-renewal of ES cells, they displayed limited potential for cellular reprogramming. These results indicate that the intracellular localization of Oct4, which is dependent on nucleocytoplasmic shuttling, must be more strictly regulated for cellular reprogramming, suggesting that Oct4 plays differential roles in the self-renewal of ES cells and in somatic cell reprogramming.

Generation of rat monoclonal antibody specific for mouse importin α8.

The transport of proteins in and out of the nucleus plays important roles in major cellular processes, such as signal transduction and regulation of cell cycle. Proteins that contain a nuclear localization signal (NLS) are recognized by an importin α/ß heterodimer and targeted to the nucleus. Here, we report the generation of a rat monoclonal antibody (MAb) that recognizes a novel importin α family member, importin α8, which is expressed during oocyte maturation and early embryonic development. Immunoblot and immunolocalization analyses showed that this MAb was specific for mouse importin α8 and not other importin α family members. These data suggest that this MAb is useful for analyzing molecular functions of importin α8.

Generation and characterization of a monoclonal antibody against importin α7/NPI-2.

Many nuclear proteins are transported into the nucleus via the importin α/ß-mediated pathway. Importin α comprises a multigene family. In this study, we generated and characterized a rat monoclonal antibody (MAb) 3F8 to importin α7. The antibody was generated by the hybridization of mouse myeloma cells with lymph node cells from an immunized rat. The MAb 3F8 specifically recognized importin α7 among importin α isoforms as evidenced by immunoblotting analysis. Furthermore, MAb 3F8 detected exogenous importin α7 in COS-7 cells by immunofluorescence. This MAb will be useful in the analysis of the isoform-specific function of importin α7.

Targeted disruption of one of the importin α family members leads to female functional incompetence in delivery.

Importin α mediates the nuclear import of proteins through nuclear pore complexes in eukaryotic cells, and is common to all eukaryotes. Previous reports identified at least six importin α family genes in mice. Although these isoforms show differential binding to various import cargoes in vitro, the in vivo physiological roles of these mammalian importin α isoforms remain unknown. Here, we generated and examined importin α5 knockout (impα5(-/-)) mice. These mice developed normally, and showed no gross histological abnormalities in most major organs. However, the ovary and uterus of impα5(-/-) female mice exhibited hypoplasia. Furthermore, we found that impα5(-/-) female mice had a 50% decrease in serum progesterone levels and a 57% decrease in progesterone receptor mRNA levels in the ovary. Additionally, impα5(-/-) uteruses that were treated with exogenous gonadotropins displayed hypertrophy, similarly to progesterone receptor-deficient mice. Although these mutant female mice could become pregnant, the total number of pups was significantly decreased, and some of the pups were dead at birth. These results suggest that importin α5 has essential roles in the mammalian female reproductive organs.

Mice lacking Ran binding protein 1 are viable and show male infertility.

The small GTPase Ran plays important roles in multiple aspects of cellular function. Maximal RanGAP activity is achieved with the aid of RanBP1 and/or presumably of RanBP2. Here, we show that RanBP1-knockout mice are unexpectedly viable, and exhibit male infertility due to a spermatogenesis arrest, presumably caused by down-regulation of RanBP2 during spermatogenesis. Indeed, siRNA-mediated depletion of RanBP2 caused severe cell death only in RanBP1-deficient MEFs, indicating that simultaneous depletion of RanBP1 and RanBP2 severely affects normal cell viability. Collectively, we conclude that the dramatic decrease in "RanBP" activity impairs germ cell viability and affects spermatogenesis decisively in RanBP1-knockout mice.

Specific monoclonal antibody against the nuclear pore complex protein, Nup96.

Nup96 is a component of the Nup107-160 complex, the largest subunit of the nuclear pore complex. Nup96 is generated as a precursor protein with Nup98. However, the mechanism by which Nup96 contributes to cell function is not clear. We report here on the preparation of a monoclonal antibody (MAb) directed against mouse Nup96. The antibody was produced by the hybridization of mouse myeloma cells with lymph node cells from an immunized rat. The antibody, MAb 4H5, specifically recognized Nup96, as evidenced by immunoblotting using the whole cell lysates. In immunostaining using MAb 4H5, a nuclear rim staining pattern was observed. This antibody will be useful in immunoblotting and immunolocalization experiments, as well as further analyses of the biological function and cellular dynamics of this protein.

Dual-ligand effect of transferrin and transforming growth factor alpha on polyethyleneimine-mediated gene delivery.

In order to enhance the internalization of exogenous gene and add cell specificity to non-viral vectors, receptor-binding elements have been widely utilized to mimic the virus infection. Herein, for the purpose of intensifying the effects of the ligand on gene delivery, dual receptor-binding elements, transferrin (Tf) and transforming growth factor alpha (TGFalpha), were introduced into the polyethyleneimine polyplex. The transfection and internalization efficiency by dual Tf- and TGFalpha-introduced polyplex (Tf&TGFalpha-polyplex) was examined in A549 and CHO-K1 cells, respectively. In A549, Tf&TGFalpha-polyplex had higher transfection efficiency when compared to that by single Tf- or TGFalpha-introduced polyplex (Tf-polyplex and TGFalpha-polyplex), respectively, while no enhancement was observed in CHO-K1. Moreover, in A549, the internalization efficiency of dual Tf&TGFalpha-polyplex was higher than that of single Tf- and TGFalpha-polyplex. In contrast, in CHO-K1, no difference in internalization efficiency was observed. In the presence of excess free transferrin or TGFalpha, the internalization efficiency of Tf&TGFalpha-polyplex was strongly inhibited only in A549, not in CHO-K1. In summary, the enhancement of internalization efficiency by dual ligands is an important factor for improving transfection efficiency. In addition, the effect of dual ligands depends on cell species; receptor-mediated and efficient internalization may be related to this enhanced transfection efficiency.