Oncogenic nucleoporin CAN/Nup214 interacts with vitamin D receptor and modulates its function.

Vitamin D receptor (VDR) is a ligand-dependent transcription factor and should be located in nucleus to transactivate target genes. To explore the molecules that interact with VDR and facilitate its nuclear localization, we screened a human kidney cDNA library using the yeast two-hybrid approach, and found that VDR binds to the carboxy-terminal region of an oncogenic nucleoporin, CAN/Nup214. CAN/Nup214 was originally identified through its involvement in a certain type of acute myeloid leukemia, and is a component of nuclear pore complex (NPC). Co-immunoprecipitation experiments confirmed the interaction between VDR and the carboxy-terminus of CAN/Nup214 containing a cluster of the phenylalanine-glycine (FG) repeat in mammalian cells. The exogenously expressed full-length CAN/Nup214 was localized predominantly at the nuclear envelope, suggesting its integration in the NPCs. We then examined the effects of exogenous expression of full-length CAN/Nup214 and its carboxy-terminal fragment on the VDR-mediated transactivation. The overexpression of full-length CAN/Nup214 facilitated the VDR-mediated transactivation, while the expression of the carboxy-terminal fragment suppressed it. The DNA-binding domain of VDR was required for the facilitation of the VDR-dependent transactivation by CAN/Nup214. Although the subcellular distribution of VDR was not obviously altered by the overexpression of full-length CAN/Nup214 or the carboxy-terminal fragment, the expression of the carboxy-terminal fragment inhibited the interaction between full-length CAN/Nup214 and VDR. These results indicate that CAN/Nup214 interacts with VDR and modulates its function as a transcription factor.

Generation and characterization of a monoclonal antibody against NPI-1 subfamily of importin alpha.

Most karyophilic proteins are transported into the nucleus through the importin-mediated pathway. Importin alpha acts as a receptor for classical nuclear localization signal (NLS)-containing proteins. At present, the existence of several isoforms of importin alpha in mammals is known. In this study we report on the generation of a rat monoclonal antibody (MAb) 2D9 to importin alpha NPI-1 subfamily members (importin alpha5/NPI-1 and importin alpha7/S2) using the rat lymph node method and the characterization of this antibody. In several different cultured cell extracts, MAb 2D9 reacted to endogenous NPI-1 subfamily in Western blotting experiments. Epitope mapping using recombinant deletion mutants indicated that MAb 2D9 recognized arm motif in importin alpha5/NPI-1. Using immunofluorescence, MAb 2D9 detected NPI-1 subfamily in the cytoplasm of HeLa cells. Moreover, endogenous NPI-1 subfamily was dominantly localized in the nuclei of H(2)O(2)-treated HeLa cells, suggesting that NPI-1 subfamily accumulates in the nucleus in response to oxidative stress, like importin alpha1/Rch1.

Lrp6 hypomorphic mutation affects bone mass through bone resorption in mice and impairs interaction with Mesd.

Low-density lipoprotein receptor-related protein 5 (LRP5) regulates bone acquisition by controlling bone formation. Because roles of LRP6, another co-receptor for Wnts, in postnatal bone metabolism have not been fully elucidated, we studied bone phenotype in mice harboring an Lrp6 hypomorphic mutation, ringelschwanz (rs), and characterized the mutant protein. First, we performed pQCT, bone histomorphometry, and immunohistochemistry on tibias of Lrp6(rs/rs) and Lrp6(+/+) mice and determined biochemical parameters for bone turnover. Lrp6(rs/rs) mice exhibited reduced trabecular BMD in pQCT. Bone histomorphometry showed low bone volume and decreased trabecular number, which were associated with increased eroded surface. Urinary deoxypyridinoline excretion was increased in Lrp6(rs/rs) mice, whereas levels of serum osteocalcin were comparable between Lrp6(rs/rs) mice and wildtype littermates. Increase in cell number and mineralization of calvariae-derived osteoblasts were not impaired in Lrp6(rs/rs) osteoblasts. Rankl expression was increased in Lrp6(rs/rs) osteoblasts both in vivo and in vitro, and osteoclastogenesis and bone-resorbing activity in vitro were accelerated in Lrp6(rs/rs) cells. Treatment with canonical Wnt suppressed Rankl expression in both in primary osteoblasts and ST2 cells. Overexpression of Lrp6 also suppressed Rankl expression, whereas the Lrp6 rs mutant protein did not. Functional analyses of the Lrp6 rs mutant showed decreased targeting to plasma membrane because of reduced interaction with Mesoderm development (Mesd), a chaperone for Lrp6, leading to impaired Wnt/beta-catenin signaling. These results indicate that Lrp6-mediated signaling controls postnatal bone mass, at least partly through the regulation of bone resorption. It is also suggested that the interaction with Mesd is critical for Lrp6 to function.

Functional analysis of nuclear pore complex protein Nup62/p62 using monoclonal antibodies.

The nuclear pore complex (NPC) is an enormous structure embedded in the double membrane of the nuclear envelope that acts as a passageway for nucleocytoplasmic transport. The vertebrate NPC is comprised of about 30 unique proteins. Nup62/p62, a major component of the NPC, has been reported to interact directly with several nuclear transport factors, including importin-beta and NTF2. However, it has not been shown how the interaction of Nup62/p62 with transport factors is involved in nucleocytoplasmic transport. The present study reports on the preparation of monoclonal antibodies (MAbs) directed against human Nup62/p62 and a functional analysis of Nup62/p62 using antibodies in living cells. Hybridomas producing the antibodies were produced by the hybridization of mouse myeloma cells with medial iliac lymph node cells from an immunized rat. These MAbs specifically recognized Nup62/p62 as evidenced by immunoblotting analysis using a nuclear membrane fraction. In the immunostaining using MAbs, a punctuate nuclear rim staining pattern was observed. Moreover, cytoplasmic injected-anti-Nup62/p62 MAbs were rapidly targeted to the nuclear pore of cultured cells and some of them inhibited normal cell division, causing the formation of abnormal nuclei. The antibodies described in this study provide the means for immunochemical analyses of the NPC protein Nup62/p62 in mammalian cells, and represent useful molecular tools that should permit a better understanding of the biological roles and cellular dynamics of this protein in nucleocytoplasmic transport, cell division, and nuclear organization.

Importin 4 is responsible for ligand-independent nuclear translocation of vitamin D receptor.

Vitamin D receptor (VDR) is localized in nuclei and acts as a ligand-dependent transcription factor. To clarify the molecular mechanisms underlying the nuclear translocation of VDR, we utilized an in vitro nuclear transport assay using digitonin-permeabilized semi-intact cells. In this assay, recombinant whole VDR-(4-427) and a truncated mutant VDR-(4-232) lacking the carboxyl terminus of VDR were imported to nuclei even in the absence of ligand. In contrast, VDR-(91-427) lacking the amino-terminal DNA-binding domain was not imported to nuclei in the absence of ligand, and was efficiently imported in its liganded form. These results suggested that there are two distinct mechanisms underlying the nuclear transport of VDR; ligand-dependent and -independent pathways, and that the different regions of VDR are responsible for these processes. Therefore, we performed the yeast two-hybrid screening using VDR-(4-232) as the bait to explore the molecules responsible for ligand-independent nuclear translocation of VDR, and have identified importin 4 as an interacting protein. In the reconstruction experiments where transport factors were applied as recombinant proteins, recombinant importin 4 facilitated nuclear translocation of VDR regardless of its ligand, whereas importin beta failed in transporting VDR even in the presence of ligand. In conclusion, importin 4, not importin beta, is responsible for the ligand-independent nuclear translocation of VDR.

Extracellular signal-dependent nuclear import of STAT3 is mediated by various importin alphas.

The signal transducer and activator of transcription 3 (STAT3) is a transcription factor that is involved in a variety of biological functions. STAT3 is activated by cytokines and growth factors via the phosphorylation of a tyrosine residue, dimerization, and subsequent nuclear translocation. However, the mechanism of its nuclear translocation is unclear. A study of the cytokine-stimulated import of STAT3 into the nucleus is reported herein. An oncostatin M (OSM)-dependent nuclear import assay system was first established in living cells. Using this system, we demonstrated that the microinjection of the importin alpha5/NPI-1 mutant, an anti-importin beta antibody, and the RanQ69L mutant inhibited the nuclear import of STAT3. Second, we showed that tyrosine-phosphorylated STAT3 associates, not only with importin alpha5/NPI-1 but also with other importin alphas, as a result of OSM stimulation, as evidenced by a solution binding assay. These findings suggest that the extracellular signal-dependent nuclear transport of STAT3 is mediated by various importin alphas, importin beta, and Ran.

Specific monoclonal antibody against nuclear import factor, importin alpha1/Rch1.

Importin alpha1/Rch1, an importin alpha family member, mediates the nuclear import of karyophilic proteins. The present study reports on a monoclonal antibody (MAb) directed against mammalian importin alpha1/Rch1, which was produced by the hybridization of mouse myeloma cells with lymph node cells of an immunized rat. The MAb 1A6 specifically recognized importin alpha1/Rch1 among the importin alpha isoforms, as evidenced by Western blotting. Furthermore, 1A6 detected importin alpha1/Rch1 in HeLa cells by immunofluorescence. This MAb will be useful in immunolocalization and immunoblotting experiments, conducted on different tissue types, to determine the levels of expression of importin alpha1/Rch1 throughout development, as well as further analyses of the biological function and cellular dynamics of this protein.

Generation of a rat monoclonal antibody specific for importin alpha3/Qip1.

Importin alpha, which mediates the nuclear import of nuclear localization signal (NLS)-containing proteins, is a member of nuclear transport factors. Importin alpha binds directly NLS and functions as an adapter for accessing the importin beta-dependent import pathway. To date, several isoforms of importin alpha have been identified and classified into three subfamilies in higher eukaryotes. In this study, we report on the production of a rat monoclonal antibody (MAb) against importin alpha3/Qip1, a member of the importin alpha family, using a rat medial iliac lymph node method. The MAb 3D10 produced, reacted with both recombinant and endogenous importin alpha 3/Qip1. Immunoblotting analysis revealed that MAb 3D10 exclusively recognizes importin alpha3/Qip1 among members of the importin alpha family, in various mammalian cells.