Elsevier

Gene

Volume 255, Issue 1, 5 September 2000, Pages 105-116
Gene

Genomic organization, chromosomal localization and regulation of expression of the neuronal nuclear matrix protein NRP/B in human brain tumors

https://doi.org/10.1016/S0378-1119(00)00297-3Get rights and content

Abstract

The nuclear matrix and its role in cell physiology are largely unknown, and the discovery of any matrix constituent whose expression is tissue- and/or cell-specific offers a new avenue of exploration. Studies of the novel neuronal nuclear matrix protein, NRP/B, reveal that it is an early and highly specific marker of neuronal induction and development in vertebrates, since its expression is restricted mainly to the developing and mature nervous system. These studies also show that NRP/B is involved in neuronal differentiation. To further examine the structure–function of NRP/B, we have cloned and characterized the murine Nrp/b gene. The murine gene consists of four exons interrupted by three introns that span 7.6 kb of DNA. The complete open reading frame is localized in exon 3, suggesting that NRP/B is highly conserved during evolution. Chromosomal analysis shows that NRP/B is localized to chromosome 13 in mouse and chromosome 5q12–13 in human.

Since our previous studies demonstrated that NRP/B is expressed in primary hippocampal neurons but not in primary astrocytes, we have characterized NRP/B mRNA and protein expression in various brain cell lines and in human brain tumors. Abundant expression of NRP/B mRNA and protein was observed in human neuroblastoma cell lines (IMR32, SKN-MC, SKN-SH), in glioblastoma cell lines (A172, T98G, U87-MG, U118-MG, U138-MG, and U373-MG), in neuroglioma (H4) and astrocytoma cell lines (CCF-STTG1 and SW1088). Confocal analysis of NRP/B in U87-MG glioblastoma cells indicated nuclear localization of NRP/B. NRP/B expression was also observed in human primary brain tumors including glioblastoma multiformae and astrocytomas (total of five cases). These results suggest that NRP/B expression is upregulated in human brain tumors including glioblastomas and astrocytomas, while under normal conditions NRP/B expression is restricted to neurons. This study implicates a role for NRP/B in brain tumor development.

Introduction

The nuclear matrix constitutes the three-dimensional filamentous protein network that maintains the domain organization of the nucleus. This component of the nuclear architecture provides the internal scaffold of the nucleus. It is formed by an ordered and highly compartmentalized protein structure consisting of a nuclear lamina, a residual nucleolus, and an internal matrix composed of a non-chromatin fibrogranular network associated with DNA (Berezney et al., 1996, Stein and Berezney, 1996). The nuclear matrix has been implicated in transcription, regulation of gene expression, the cell cycle, primary transcription processing and in linkages to intermediate filaments of the cytoskeleton (He et al., 1995, Loidl and Eberharter, 1995).

A cellular hallmark of the transformed phenotype is an altered nuclear shape and the presence of abnormal nucleoli. Structural alterations of the nucleus are prevalent in cancer cells and are commonly used as pathological markers of transformation in many types of cancer. Nuclear shape is thought to reflect the internal nuclear structure and is determined, at least in part, by the nuclear matrix (Dworetzky et al., 1990, Peinta et al., 1989). The nuclear matrix contains a number of associated proteins that have been found to be involved in malignant transformation (Fey and Penman, 1984, Getzenberg et al., 1996, Keese et al., 1994). It has been directly or indirectly implicated in most of the changes considered to be pathologic hallmarks of malignant transformation, such as alterations in DNA ploidy, DNA content, nuclear shape and proliferative states (Berezney and Jeon, 1995).

Ataxin-1 (the protein encoded by the SCA1 gene), which is involved in the neurodegenerative disorder spinocerebellar ataxia, alters nuclear-matrix-associated structures (Skinner et al., 1997). Despite the apparent importance of the nuclear matrix in the regulation of many biological processes, its roles in cell physiology and in neuronal differentiation are largely unknown.

Recently, we have discovered and characterized a novel nuclear matrix protein, NRP/B (nuclear restricted protein/brain), which contains two major structural elements: a BTB domain-like structure in the predicted N-terminus, and a ‘kelch motif’ in the predicted C-terminal domain (Kim et al., 1998). NRP/B mRNA (5.5 kb) is expressed predominantly in human fetal and adult brain, with minor expression in kidney and pancreas. During mouse embryogenesis, NRP/B mRNA expression is upregulated in the nervous system. ENC-1, a mammalian kelch-related gene that is specifically expressed in the nervous system, was also reported to be the murine homolog of NRP/B (Hernandez et al., 1997, Kim et al., 1998). NRP/B/ENC-1 is expressed in the prospective neuroectodermal region of the epiblast during early gastrulation and throughout the nervous system later in development. NRP/B/ENC-1 expression is highly dynamic and, after neurulation, preferentially defines prospective cortical areas. Expression of NRP/B/ENC-1 was detected at the preneurulation stage of the mouse embryo (E 6.5) in the prospective neuroectodermal region of the epiblast that later differentiates predominantly into neuroectodermal cells (Hernandez et al., 1997, Kim et al., 1998). No expression of NRP/B/ENC-1 was detected in any extraembryonic tissue. At E 8.0, its expression was detected in ectodermal derivatives and continued to be strongly expressed in the hippocampus and neocortex (Hernandez et al., 1997, Kim et al., 1998).

The BTB/POZ domain at the N-terminus of NRP/B consists of approx. 115 amino acids and is expressed in several members of the kelch family (Xue and Cooley, 1993). This domain, found primarily in zinc finger proteins, defines a newly characterized protein–protein interaction interface (Bardwell and Treisman, 1994), and also mediates both dimer and heterodimer formation in vitro (Albagli et al., 1995). NRP/B also shares significant homology to the ‘kelch’ repeats found in several kelch-related genes (von Bulow et al., 1995, Way et al., 1995, Xue and Cooley, 1993) and in a large number of open reading frames (ORFs) within the genome of poxviruses (von Bulow et al., 1995, Way et al., 1995). NRP/B contains six repeats of kelch in the C-terminal half of the protein, while each repeat consists of about 50 amino acids. These motifs may have functional significance in binding actin, protein folding or in protein–protein interactions.

We have shown previously that NRP/B protein is expressed in rat primary hippocampal neurons, but not in primary astrocytes, and that its expression is upregulated during the differentiation of rat PC12 cells, murine Neuro-2A and human SH-SY5Y neuroblastoma cells (Kim et al., 1998). Overexpression of NRP/B in these cells augmented neuronal process formation, while treatment with antisense NRP/B oligodeoxynucleotides inhibited the neurite development of rat primary hippocampal neurons as well as neuronal process formation during differentiation of PC12 cells.

In this study, we aimed to characterize the genomic organization, chromosomal localization and expression of NRP/B in brain tumor cell lines and human primary brain tumors. We have determined the chromosomal assignment of the human NRP/B gene by analysis of somatic cell hybrids, and we have mapped the mouse NRP/B gene by backcross analysis. Our results suggest that NRP/B expression is upregulated in brain tumors.

Section snippets

Materials

Chemical reagents were purchased from Sigma (St. Louis, MO). The murine fetal λ-gt10 cDNA library was obtained from Dr. Kunkle, Children's Hospital, Boston, MA. Restriction endonucleases, modifying enzymes, terminal deoxynucleotidyl transferase, random priming kits, and Sephadex G-25 quickspin columns were purchased from Pharmacia-LKB (Piscataway, NJ) and New England BioLabs (Beverly, MA). The primers for polymerase chain reaction (PCR), RT–PCR and sequencing were synthesized using an automated

Cloning, sequence analysis, and genomic organization of the murine Nrp/b gene

We previously observed that Nrp/b is expressed in rat primary hippocampal neurons but not in astrocytes, and that its level of expression is upregulated during neuronal differentiation (Kim et al., 1998). To determine the exon–intron organization of the murine Nrp/b gene and to identify its potential tissue-specific response elements, we screened approx. 6×105 total recombinants, from a murine liver genomic library (γ-EMBL-3), for genomic clones under conditions of high stringency with a 260 bp

Discussion

In this study, we describe the genomic organization of the Nrp/b gene, and the chromosomal localization and expression of NRP/B protein in human primary brain tumors. NRP/B is a novel nuclear matrix protein (Kim et al., 1998), which is expressed abundantly in the brain and appears to be localized in primary neuronal cells. NRP/B contains a BTB/POZ domain in the N-terminus and ‘kelch’ repeats at the C-terminus, and appears to play an important role in neuronal differentiation. Based on our

Acknowledgements

We thank Dr. Bijia Deng for her help in establishing rat primary hippocampal cultures. The authors wish to thank Lucy Rowe and Ed Birkenmeier of The Jackson Laboratory for supplying the DNA panel and for performing the analyses of linkage data. We thank Dr. Hava Avraham for her advice and comments on the manuscript, Janet Delahanty for her help in editing the manuscript, Dan Kelley for preparing the artwork, and Mikyung Kim for her typing assistance. This work was supported by grants from the

References (30)

  • Z. Chen et al.

    Fusion between a novel Kruppel-like zinc finger gene and the retinoic acid receptor-alpha locus due to a variant t(11,17) translocation associated with acute promyelocytic leukaemia

    EMBO J.

    (1993)
  • P. Dhordain et al.

    Corepressor SMRT binds the BTB/POZ repressing domain of the LAZ3/BCL6 oncoprotein

    Proc. Natl. Acad. Sci. USA

    (1997)
  • S.I. Dworetzky et al.

    Progressive changes in the protein composition of the nuclear matrix during rat osteoblast differentiation

    Proc. Natl. Acad. Sci. USA

    (1990)
  • E.G. Fey et al.

    Tumor promoters induce a specific morphological signature in the nuclear matrix-intermediate filament scaffold of Madin–Darby canine kidney (MDCK) cell colonies

    Proc. Natl. Acad. Sci. USA

    (1984)
  • R.H. Getzenberg et al.

    Bladder cancer-associated nuclear matrix proteins

    Cancer Res.

    (1996)
  • Cited by (38)

    • Kelch-like proteins: Physiological functions and relationships with diseases

      2019, Pharmacological Research
      Citation Excerpt :

      Recently, a study demonstrated that KLHL22 interacts with the DEP domain containing 5 (DEPDC5)-mediated mammalian target of rapamycin 1 (mTORC1) signalling pathway, that KLHL22 mRNA and protein levels are markedly elevated in breast tumours compared with adjacent normal tissues, and that depletion of KLHL22 could suppress tumour growth in nude mice, suggesting that KLHL22 is a potential oncogene in breast cancer [81]. ( 5) KLHL37, also known as ectodermal-neural cortex 1 (ENC1), is an early and specific marker of neurons during the development of the central nervous system, whose gene mutations may give rise to brain tumours including glioblastomas and astrocytomas [82] by enhancing cell proliferation, blocking apoptosis and influencing nuclear cytoskeleton dynamics [19]. Additionally, upregulation of ENC1 expression confers the risk of colorectal carcinomas [83] and hairy cell leukaemia (HCL) [84], and it is also significantly associated with the prognosis of patients with ovarian cancer [85].

    • Neonatal ventral hippocampus lesion changes nuclear restricted protein/brain (NRP/B) expression in hippocampus, cortex and striatum in developmental periods of rats

      2016, Neuroscience
      Citation Excerpt :

      During gastrulation, NRP/B is expressed in the prospective neuroectodermal region of the epiblast, and its expression is maintained in the late developmental phase. Kim’s studies showed NRP/B was expressed in the primary hippocampal neurons, but not in primary astrocytes and abundant expression of NRP/B mRNA and protein was observed in human neuroblastoma cell lines (IMR32, SKN-MC, SKN-SH), in glioblastoma cell lines (A172, T98G, U87-MG, U118-MG, U138-MG, and U373-MG), in neuroglioma (H4) and astrocytoma cell lines (CCF-STTG1 and SW1088) (Kim et al., 1998, 2000). Studies in vitro showed NRP/B was involved in neuronal differentiation and the BTB domain of NRP/B is essential for neurite outgrowth (Kim et al., 1998, 2005), indicating that NRP/B is critical for the normal development of the neuronal system.

    View all citing articles on Scopus

    This paper is dedicated to Raphael Recanati and his family for their friendship and support for our research program.

    1

    Both authors contributed equally to this work.

    View full text