Yayoi Kinoshita

Purα Is Essential for Postnatal Brain Development and Developmentally Coupled Cellular Proliferation As Revealed by Genetic Inactivation in the Mouse

ABSTRACT The single-stranded DNA- and RNA-binding protein, Purα, has been implicated in many biological processes, including control of transcription of multiple genes, initiation of DNA replication, and RNA transport and translation. Deletions of the PURA gene are frequent in acute myeloid leukemia. Mice with targeted disruption of the PURA gene in both alleles appear normal at birth, but at 2 weeks of age, they develop neurological problems manifest by severe tremor and spontaneous seizures and they die by 4 weeks. There are severely lower numbers of neurons in regions of the hippocampus and cerebellum of PURA−/− mice versus those of age-matched +/+ littermates, and lamination of these regions is aberrant at time of death. Immunohistochemical analysis of MCM7, a protein marker for DNA replication, reveals a lack of proliferation of precursor cells in these regions in the PURA−/− mice. Levels of proliferation were also absent or low in several other tissues of the PURA−/− mice, including those of myeloid lineage, whereas those of PURA+/− mice were intermediate. Evaluation of brain sections indicates a reduction in myelin and glial fibrillary acidic protein labeling in oligodendrocytes and astrocytes, respectively, indicating pathological development of these cells. At postnatal day 5, a critical time for cerebellar development, Purα and Cdk5 were both at peak levels in bodies and dendrites of Purkinje cells of PURA+/+ mice, but both were absent in dendrites of PURA−/− mice. Purα and Cdk5 can be coimmunoprecipitated from brain lysates of PURA+/+ mice. Immunohistochemical studies reveal a dramatic reduction in the level of both phosphorylated and nonphosphorylated neurofilaments in dendrites of the Purkinje cell layer and of synapse formation in the hippocampus. Overall results are consistent with a role for Purα in developmentally timed DNA replication in specific cell types and also point to a newly emerging role in compartmentalized RNA transport and translation in neuronal dendrites.

Role of Purα in targeting mRNA to sites of translation in hippocampal neuronal dendrites

Using genetic inactivation in the mouse, PURA, encoding Purα, is demonstrated to be essential for developmentally‐timed dendrite formation in the cerebellum and hippocampus. Comparison of RNA species bound by Purα prompts the hypothesis that Purα functions with non‐coding RNA in transport of certain mRNA molecules to sites of translation in dendrites. Purα binds to human BC200 RNA, implicated in dendritic targeting, and this has homologies to 7SL RNA, implicated in compartmentalized translation. Results using hippocampal rat neurons in situ show that Purα binds to BC1 RNA, implicated in dendritic targeting as a mouse counterpart of BC200, and to mRNA molecules translated in dendrites; Purα is specifically located in dendrites, where it is colocalized with Map2, but not in axons, where it fails to colocalize with Ankyrin G. Purα and Staufen are colocalized at dendritic sites of mRNA translation. Microtubule disruptors inhibit Purα dendritic targeting and allow its mislocalization to axons. Using mouse brain, double‐RNA immunoprecipitation places Purα together with Staufen or FMRP on BC1 RNA and specific mRNA species in vivo. These results help define a mechanism by which Purα targets specific mRNA molecules to sites of dendritic translation.

Site-specific Loading of an MCM Protein Complex in a DNA Replication Initiation Zone Upstream of the c-MYC Gene in the HeLa Cell Cycle

The MCM proteins participate in an orderly association, beginning with the origin recognition complex, that culminates in the initiation of chromosomal DNA replication. Among these, MCM proteins 4, 6, and 7 constitute a subcomplex that reportedly possesses DNA helicase activity. Little is known about DNA sequences initially bound by these MCM proteins or about their cell cycle distribution in the chromatin. We have determined the locations of certain MCM and associated proteins by chromatin immunoprecipitation (ChIP) in a zone of initiation of DNA replication upstream of the c-MYC gene in the HeLa cell cycle. MCM7 and its clamp-loading partner Cdc6 are highly specifically colocalized by ChIP and re-ChIP in G1 and early S on a 198-bp segment located near the center of the initiation zone. ChIP and Re-ChIP colocalizes MCM7 and ORC1 to the same segment specifically in late G1. MCM proteins 6 and 7 can be coimmunoprecipitated throughout the cell cycle, whereas MCM4 is reduced in the complex in late S and G2, reappearing upon mitosis. MCM7 is not visualized by immunohistochemistry on metaphase chromosomes. MCM7 is recruited to multiple sites in chromatin in S and G2, at which time it is not detected with ORC1. The rate of dissemination is surprisingly slow and is unlikely to be simply attributed to progression with replication forks. Results indicate sequence-specific loading of MCM proteins onto DNA in late G1 followed by a recruitment to multiple sites in chromatin subsequent to replication.

SMAD proteins of oligodendroglial cells regulate transcription of JC virus early and late genes coordinately with the Tat protein of human immunodeficiency virus type 1

JC virus (JCV) is the aetiological agent of progressive multifocal leukoencephalopathy (PML), a fatal, demyelinating disease of the brain affecting people with AIDS. Although immunosuppression is involved in infection of the brain by JCV, a direct influence of human immunodeficiency virus type 1 (HIV-1) has also been established. The Tat protein of HIV-1 has been implicated in activation of the cytokine transforming growth factor (TGF)-beta in HIV-1-infected cells and in stimulating JCV gene transcription and DNA replication in oligodendroglia, the primary central nervous system cell type infected by JCV in PML. This study demonstrated that Tat can cooperate with SMAD proteins, the intracellular effectors of TGF-beta, at the JCV DNA control region (CR) to stimulate JCV gene transcription. Tat stimulated JCV early gene transcription in KG-1 oligodendroglial cells when expressed via transfection or added exogenously. Using chromatin immunoprecipitation, it was shown that exogenous Tat enhanced binding of SMAD2, -3 and -4 and their binding partner Fast1 to the JCV CR in living cells. When SMAD2, -3 and -4 were expressed together, Tat, expressed from plasmid pTat, stimulated transcription from both early and late gene promoters, with the early promoter exhibiting stimulation of >100-fold. Tat, SMAD4 and JCV large T-antigen were all visualized in oligodendroglial cells at the border of an active PML lesion in the cerebral frontal lobe. These results revealed a positive reinforcement system in which the SMAD mediators of the TGF-beta system act cooperatively with Tat to stimulate JCV gene transcription.

gC1qR Expression in Normal and Pathologic Human Tissues Differential Expression in Tissues of Epithelial and Mesenchymal Origin

The gC1qR (i.e., gC1q receptor, gC1q binding protein, p32, p33) is a multifunctional cellular protein that interacts with components of the complement, kinin, and coagulation cascades and select microbial pathogens. Enhanced gC1qR expression has been reported in adenocarcinomas arising in a variety of organs. The present study compared gC1qR expression in normal, inflammatory, dysplastic, and malignant tissue of epithelial and mesenchymal origin. gC1qR expression was visualized in tissue sections by immunohistochemistry using the 60.11 monoclonal antibody (i.e., IgG1 mouse monoclonal antibody directed against gC1qR) and the UltraVision LP Detection System. Sections were counterstained with hematoxylin and examined by light microscopy. Strongest gC1qR expression was noted in epithelial tumors of breast, prostate, liver, lung, and colon, as well as in squamous and basal cell carcinoma of the skin. However, increased gC1qR staining was appreciated also in inflammatory and proliferative lesions of the same cell types, as well as in normal continuously dividing cells. In contrast, tumors of mesenchymal origin generally stained weakly, with the exception of osteoblasts, which stained in both benign and malignant tissues. The data suggest that increased gC1qR expression may be a marker of benign and pathologic cell proliferation, particularly in cells of epithelial origin, with potential diagnostic and therapeutic applications.

Alterations in Nuclear Pore Architecture Allow Cancer Cell Entry into or Exit from Drug-Resistant Dormancy

Phenotypic diversity arises in tumors just as it does in developing organisms, and tumor recurrence frequently manifests from the selective survival of divergent drug-resistant cells. Although the expanding tumor cell population may be successfully targeted, drug-resistant cells may persist and sustain the tumor or enter dormancy before igniting a future relapse. Herein, we show that partial knockdown of nucleoporin p62 (NUP62) by small-interfering RNA confers cisplatin resistance to cultured high-grade ovarian carcinoma cells. Treatment with NUP62 small-interfering RNA and cisplatin leaves resistant cells in a state of dormancy; some dormant cells can be induced to proliferate by transient induction of NUP62 expression from an ectopic expression construct. In addition to suggesting functional links between nuclear pore complex architecture and cancer cell survival, the culture system provides a novel experimental window into the dynamics of tumor cell drug resistance and dormancy.

Role for NUP62 depletion and PYK2 redistribution in dendritic retraction resulting from chronic stress.

Significance Associated with depression and cognitive impairment, chronic stress causes reversible dendritic shrinkage particularly evident in the hippocampal CA3 region in several animal models. In further elucidating the molecular events leading to dendritic shrinkage, this study reveals two unexpected mechanisms reduction in translated transcripts of a nuclear pore complex protein, nucleoporin p62 (NUP62), and tyrosine phosphorylation of NUP62, that appear to act cumulatively in chronic stress to reduce NUP62 content in CA3 neurons. Subcellular redistribution of activated proline-rich tyrosine kinase 2 in chronically stressed pyramidal neurons suggests a mechanism for stress-induced tyrosine phosphorylation of NUP62. Furthermore, evidence from cultured hippocampal neurons shows that diminishing the content of NUP62, which functions in nucleocytoplasmic transport and chromatin organization, results in simplification and shortening of dendritic arbors. Genetic evidence suggests cell-type–specific functions for certain nucleoporins, and gene expression profiling has revealed that nucleoporin p62 (NUP62) transcripts are decreased in the prefrontal cortex of major depressives. Chronic stress, which can precipitate depression, induces changes in the architecture and plasticity of apical dendrites that are particularly evident in the CA3 region of the hippocampus. Genetically targeted translating ribosome affinity purification revealed a selective reduction in translated Nup62 transcripts in CA3 of chronically stressed mice, and the Nup62 protein content of nuclei extracted from whole hippocampus was found to be decreased in chronically stressed rats. In cultured cells, phosphorylation of a FAK/proline-rich tyrosine kinase 2 (PYK2) consensus site in the alpha-helical domain of NUP62 (human Y422) is shown to be associated with shedding of NUP62 from the nuclear pore complex (NPC) and/or retention of NUP62 in the cytoplasm. Increased levels of phospho-Y425 Nup62 were observed in cytoplasmic fractions of hippocampi from chronically stressed rats, and immunofluorescence microscopy revealed redistribution of activated Pyk2 to the perinuclear region of stressed pyramidal neurons. Depletion of Nup62 from cultured embryonic day 18 rat hippocampal and cortical neurons resulted in simplification and retraction of dendritic arbors, without disruption of axon initial segment integrity. Thus, at least two types of mechanisms—one affecting expression and the other association with the NPC—could contribute to loss of NUP62 from CA3 pyramidal neurons during chronic stress. Their combined actions may account for the enhanced responsiveness of CA3 apical dendrites to chronic stress and may either be pathogenic or serve to protect CA3 neurons from permanent damage.

Promyelocytic leukemia zinc finger and histone H1.5 differentially stain low- and high-grade pulmonary neuroendocrine tumors: a pilot immunohistochemical study

Promyelocytic leukemia zinc finger is a zinc finger transcription factor that functions as a transcriptional repressor. Its expression has been shown to be down-regulated in hematopoietic, melanocytic, and mesothelial malignancies. Histone H1.5 is a variant of histone H1, a family of linker proteins that organizes chromosomes into higher order structures. Its function is of key importance in gene expression and has been linked to more aggressive forms of prostatic carcinoma. This study aimed to investigate the immunohistochemical detectability of promyelocytic leukemia zinc finger and histone H1.5 in pulmonary neuroendocrine tumors, comprising 11 carcinoid tumorlets, 24 typical carcinoids, 12 atypical carcinoids, 20 small cell carcinomas, 11 large cell neuroendocrine carcinomas, and 2 combined small cell carcinomas-large cell neuroendocrine carcinomas. Promyelocytic leukemia zinc finger immunohistochemistry revealed moderate or strong nuclear staining in all carcinoid tumorlets, 23 of 24 typical carcinoids, and 7 of 12 atypical carcinoids in contrast to 9 of 11 large cell neuroendocrine carcinomas, all small cell carcinoma, and both combined small cell carcinoma-large cell neuroendocrine carcinomas, which showed no nuclear immunoreactivity. Histone H1.5 immunohistochemistry revealed only focal or no immunoreactivity in all carcinoid tumorlets and 19 of 24 typical carcinoids, whereas 7 of 12 atypical carcinoids, 19 of 20 small cell carcinomas, 10 of 11 large cell neuroendocrine carcinomas, and both combined small cell carcinomas-large cell neuroendocrine carcinomas displayed positive (≥ 10%) nuclear immunoreactivity-ranging from a minority of weak staining to a majority of strong staining cases. Our data suggest that the relative expression ratios of promyelocytic leukemia zinc finger and histone H1.5 may correlate with grade of pulmonary neuroendocrine tumors. Immunohistochemical stains for these markers, especially on small biopsies with crush artifact, may prove to be diagnostically useful.

Nuclear Distributions of NUP62 and NUP214 Suggest Architectural Diversity and Spatial Patterning among Nuclear Pore Complexes

The shape of nuclei in many adherent cultured cells approximates an oblate ellipsoid, with contralateral flattened surfaces facing the culture plate or the medium. Observations of cultured cell nuclei from orthogonal perspectives revealed that nucleoporin p62 (NUP62) and nucleoporin 214 (NUP214) are differentially distributed between nuclear pore complexes on the flattened surfaces and peripheral rim of the nucleus. High resolution stimulated emission depletion (STED) immunofluorescence microscopy resolved individual NPCs, and suggested both heterogeneity and microheterogeneity in NUP62 and NUP214 immunolabeling among in NPC populations. Similar to nuclear domains and interphase chromosome territories, architectural diversity and spatial patterning of NPCs may be an intrinsic property of the nucleus that is linked to the functions and organization of underlying chromatin.

Western Blot Confirmation of the H+/K+-ATPase Proton Pump in the Human Larynx and Submandibular Gland

Objective. The authors have previously demonstrated the H+/K+-ATPase (proton pump) in human larynx and lung glands via immunohistochemistry (IHC). The present hypothesis is that the proton pump is expressed in other seromucinous glands of the digestive tract that can be confirmed by IHC and Western blot analysis. Study Design. Prospective controlled tissue analysis study. Setting. Academic medical institution. Methods. Ten anonymous fresh-frozen donor specimens were obtained, comprising 3 submandibular glands, 4 larynges, and 3 normal stomach specimens for control. Submandibular gland sections were immunostained with 2 monoclonal antibodies selectively reactive with α or β subunits of the H+/K+-ATPase. Western blot analysis was performed on all specimens. Results. Consistent IHC staining was observed in the submandibular gland specimens for both α and β subunits. Western blot analysis revealed very strong expression for the stomach at 100 kDa, corresponding to the α protein, and weak but notable banding for all larynx and submandibular gland specimens. Similar findings were noted for the 60- to 80-kDa glycosylated β subunit protein, as well as the 52-kDa β subunit precursor for all specimens. Conclusion. The H+/K+-ATPase (proton) pump is present in the human larynx and submandibular gland although in much lower concentrations than in the stomach. Proton pump involvement in human aerodigestive seromucinous glands may have a role in protecting mucosa from acid environments (local or systemic), explain heightened laryngeal sensitivity in those patients with laryngopharyngeal reflux, and be a site of action for proton pump inhibitor pharmacotherapy.