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Two Novel Genes, Human neugrin and Mouse m-neugrin, Are Upregulated with Neuronal Differentiation in Neuroblastoma Cells
Biochemical and Biophysical Research Communications 279, 526 –533 (2000) doi:10.1006/bbrc.2000.3971, available online at http://www.idealibrary.com on
Two Novel Genes, Human neugrin and Mouse m-neugrin, Are Upregulated with Neuronal Differentiation in Neuroblastoma Cells Shinsuke Ishigaki, Jun-ichi Niwa, Tsuyoshi Yoshihara, Norimasa Mitsuma, Manabu Doyu, and Gen Sobue 1 Department of Neurology, Nagoya University, School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, Aichi 466-8550, Japan
Received November 8, 2000
We herein report two new genes, human neugrin and mouse homologue m-neugrin, found by screening the cDNA library for the human spinal cord. The neugrin mRNA encodes 219 amino acids and its deduced amino acid sequence contains an NLS-like domain. No previously known motif is found in it. m-neugrin mRNA encodes 233 amino acids. Neugrin and m-Neugrin are 70% homologous in amino acid sequence. Northern analysis revealed that neugrin was strongly expressed in the heart, brain, and skeletal muscle, and m-neugrin in the liver, kidney, and brain. A transfection study indicated that these proteins are localized in the nucleus. Although the expression of neugrin was found to be ubiquitous in the nervous system, in situ hybridization showed that both neugrin and m-neugrin were expressed mainly in the neurons rather than the glial cells. Their expression was highly upregulated with the neurite outgrowth associated with neuronal differentiation in neuroblastoma cell lines. These results indicate that neugrin and m-neugrin are mainly expressed in neurons in the nervous system, and play an important role in the process of neuronal differentiation. © 2000 Academic Press
A wide variety of gene expression has been reported in association with neuronal differentiation (1– 6), and numerous stimuli, including that from retinoic acid (RA) and proteasome inhibitors, are known to cause neuronal differentiation with neurite outgrowth in neuronal cell lines (7–10). In terms of morphology, various factors act to promote neuronal differentiation, and it follows that these different factors promote neuronal differentiation on the basis of different molecular mechanisms (7, 8, 11, 12). In this report, human neuTo whom correspondence should be addressed. Fax: ⫹81-52-7442384. E-mail: [email protected]. 1
0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
grin (neurite outgrowth associated protein) and its mouse homologue, m-neugrin, new genes which are upregulated in differentiated neural cells, are characterized in terms of tissue distribution, subcellular localization, and expression profile in differentiated neuronal cells and blastomal cells. MATERIALS AND METHODS Human spinal cord cDNA library. Spinal cord was obtained at autopsy. Autopsy was performed within 6 h post mortem. Total RNA samples prepared from the anterior horn specimens were reverse transcribed by MMLV reverse transcriptase (Superscript II, Gibco BRL). Then, cDNAs were cut with BsmFI and ligated to those from a pool of 64 biotinylated adaptors cohesive to all possible overhangs. Ligated molecules were digested by BsmAI and FokI, and were recovered with streptavidin-coated paramagnetic beads. PCR was performed with the adaptor-primer and an anchored oligo-dT primer. The amplified fragments were separated by polyacrilamide gel electrophoresis. By repeating the experiment with 64 adapters, three enzymes and three anchored oligo-dT primers, 254 profiles of cDNAs were established. This procedure has been described in detail in previous reports (13, 14). Molecular cloning of neugrin. One of the cDNAs in the library described above was named neugrin, which was obtained with the 58th adapter, digested by BsmFI, and amplified by G-based anchored oligo-dT primers. The full length of the cDNA was obtained with the use of appropriate PCR based cloning methods including RACE PCR. Identification of m-neugrin. A search for murine expressed sequence tags (ESTs) identical to the neugrin fragment was made on the EST database at the National Center for Biotechnology Information (NCBI) using the BLASTN program. PCR with primers that are identical to both neugrin and the BLASTN hit EST, mouse EST No. AA815517, led to a fragment of the mouse homologue of neugrin. The full-length sequence of the gene was then determined by 5⬘ and 3⬘ RACE methods. Northern blot analysis. A sample of poly (A ⫹) RNA (2 g) was isolated from neuro2a cells or SH-SY5Y cells with respective agents (Micro-Fast Track 2.0, Invitrogen), then separated by electrophoresis through denaturing in 1% formaldehyde and transferred on to nylon membranes (Hybond N ⫹, Amersham Pharmacia Biotech). Human and mouse Multiple Tissue Northern blots were purchased from Clontech. Membranes were hybridized with a probe of neugrin, m-neugrin, or -actin, which was labeled by Alk Phos Direct (Amer-
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sham Pharmacia). The probes were obtained by denaturing the cDNA corresponding to either neugrin or m-neugrin (neugrin: 452 bp, 1307 to 1758, m-neugrin: 693 bp, 261 to 953). The -actin probe was purchased from Clontech. After overnight incubation at 55°C, membranes were washed with primary wash buffer and secondary buffer as described in the manufacturer’s specifications. The signals were then chemiluminiscently detected with CDP star (Amersham Pharmacia). The membranes were initially hybridized with the probes encoding neugrin or m-neugrin. Hybridization with the -actin probe followed as an internal control. In situ hybridization (ISH). Frozen sections (6 m thick) were prepared and fixed in 4% paraformaldehyde immediately. The deparaffinized sections were treated with proteinase K, refixed in 4% paraformaldehyde, and were prepared for in situ hybridization. Digoxigenin-labeled cRNA probes of 450 bp were generated for in situ hybridization from linearized plasmids for neugrin and m-neugrin, using Sp6 or T7 polymerase (Boehringer Mannheim). Control sense probes were also generated from each plasmid using the reciprocal RNA polymerase. Sliced tissues were hybridized for 16 h at 40°C with these cRNA probes in solution (50% formaldehyde, 10 mM Tris–HCl, pH 7.6, 1 mM EDTA, pH 8.0, 0.6 M NaCl, 0.25% SDS, 0.1 mg/ml yeast tRNA, 10% dextran sulfate, and 1 ⫻ Denhardt’s solution). The samples were then washed at room temperature in formamide/SSC (1:1) followed by 30 min at 50°C in 1 ⫻ SSC. The signals were detected immunologically with alkaline phosphatase-conjected anti-digoxigenin antibody according to the manufacturer’s protocol (Boehringer Mannheim). Expression of green fluorescent protein (GFP) fusion proteins and subcellular localization in COS-7 cells. To construct expression clones for Neugrin-GFP and m-Neugrin-GFP fusion proteins, the coding regions of neugrin and m-neugrin were amplified from human and mouse brain cDNA libraries. Restriction sites (XhoI and BamHI) for cloning into pEGFP-N1 (Clontech) were introduced by appropriate design of the PCR primers. A pEGFP-C3 (Clontech) vector without any constructs was used as a blank vector. The cloned PCR product was sequenced absolutely to exclude PCR-generated errors. Half g of each vector was preincubated with 5 l of Effectine reagent (Quiagen) diluted in 350 l medium. Transfection was performed according to the manufacturer’s protocol. The subcellular localization of GFP fusion protein in transiently transfected COS-7 cells was evaluated by microscopic study. Cell culture and differentiation. Cells were cultured in DMEM with 10% fetal calf serum (FCS) and 1% penicillin sulfate. Cells were maintained at 37°C in a saturated humidity atmosphere containing 95% air and 5% CO 2, and were seeded at an initial density of 10 4 cells per cm 2 in dishes. SH-SY5Y cells were treated in DMEM with 5% FCS and 10 M RA for 12, 24, 48, and 72 h for differentiation. Differentiation of neuro2a cells was induced in DMEM with 2% FCS and 5 M RA or DMEM with 10% FCS and 0.5 M N-carbobenzoxylLeu-Leu-leucinal (MG132), a proteasome inhibitor. Cells were harvested after incubation in medium with each agent for 12, 24, 48 and 72 h. Control cells were harvested without agents. RT-PCR analysis. Total RNA (2 g) was isolated from differentiated 2 ⫻ 10 5 neuro2a cells and SH-SY5Y cells using Micro RNA Isolation Kit (Strategene). cDNAs were synthesized by Super Script II. Then, cDNAs were amplified with specific primers for neugrin and m-neugrin. GAP-43 primers were used as a marker of neuronal differentiation. -actin primers were used as an internal control. The PCR primers used were 5⬘-GGGCATGAAGCCTCATGATCTAA-3⬘ (sense) and 5⬘-TATCCACGCAGCTCTTTCCT-3⬘ (antisense) for neugrin; 5⬘-CATGGAGCAGATCCGGTATT-3⬘ (sense) and 5⬘-TCTGAAGCTCTCGCTGATGA-3⬘ (antisense) for m-neugrin; 5⬘-AGAGCAGCCAAGCTGAAGAG-3⬘ (sense) and 5⬘-GAGGAAAGTGGACTCCCACA-3⬘ (antisense) for human GAP-43; 5⬘-CTAAGGAAAGTGCCCGACAG-3⬘; (sense) and 5⬘-TCGCCATAACAACACCAAGA-3⬘ (antisense) for mouse GAP-43; and 5⬘-CGTGCGTGACATTAAGGAGA-3⬘ (sense) and 5⬘-ACTCCTGCTTGCTGATCCAC-3⬘
(antisense) for human and mouse -actin. Each cycle consisted of denaturation at 94°C for 40 s, annealing at 60°C for 40 s, and extension at 72°C for 1 min. Appropriate numbers of PCR cycles for specific cDNAs were determined on the linear range of cycles, and also the linearity of the RT-PCR assays was ascertained with respect to the amounts of cDNA products. Aliquots of the PCR products were separated by 1% agarose gel electrophoresis and visualized by UVillumination after ethidium bromide staining.
RESULTS cDNA Cloning of neugrin and m-neugrin An 80 bp cDNA fragment was picked up from cDNA fragment profiles generated by BsmFI. The PCR primers, designed on the basis of the EST (human EST No. AA165380) that is identical for this fragment and RACE methods, yielded a specific product of 1780 bp, including a 660 bp open reading frame (ORF) (Fig. 1A, GenBank/EBI No. AB029315). A BLAST search revealed that one sequence of human DNA clone contained the entire neugrin gene (Gen Bank/EBI No. AL035467), and this clone has been mapped to 6q 12–13. A BLAST search using neugrin identified the highly homologous mouse EST (No. AA815517), and the full length of the neugrin mouse homologue of 1346 bp, including a deduced 693 bp ORF, named m-neugrin, was obtained using RACE methods (Fig. 1B). The sequence has been deposited in GenBank/EBI Data Bank under No. AB 047544. Structural Analysis The amino acid sequence alignment of both proteins shows an identity of 70%. There is an NLS-like domain from the 133rd to 136th, “RRRK” in the neugrin protein, Neugrin, although this domain changes into “QKKR” in m-Neugrin. No apparent previously known motif was found in either protein (Fig. 2). Tissue Distribution of neugrin and m-neugrin and Their Expression in the Nervous System In neugrin, Northern analysis of human MTN (Clontech) yielded a strong signal with RNA from the brain, heart, and skeletal muscle (Fig. 3A), while Northern analysis with m-neugrin exhibited relatively strong signals in the liver, kidney, heart, and brain (Fig. 3B). The pattern of neugrin mRNA expression in the human nervous system was rather ubiquitous (Fig. 3C). In human MTN, a shorter splice variant of neugrin was seen at 1.2 kbp. However, its expression level in the brain was not strong, and we investigated only the major molecule of neugrin in this study. Using mouse embryo MTN (Clontech), the expression pattern of the m-neugrin transcript in mouse embryo was examined. The level of m-neugrin mRNA increased with embryonal development (Fig. 3D).
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FIG. 1. Nucleotide and predicted amino acid sequences of neugrin (A) and m-neugrin (B). The complete sequence of neugrin (A) and m-neugrin (B) cDNA and their translation products are illustrated. The predicted amino acid sequence is shown from the first ATG codon of the open reading frame. Nucleotide and amino acid numbers are shown at the right. The GenBank Acc. No. for neugrin is AB029315. The GenBank Acc. No. for m-neugrin is AB047544.
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For further analysis of the distribution of neugrin and m-neugrin expression in the nervous system, in situ hybridization was performed using the samples of adult human postmortem spinal cords and adult mouse spinal cords. These mRNA were mainly expressed in the motor neurons, but very weak signals were also detected in the glial cells (Fig. 4). Subcellular Localization Neugrin-GFP fusion protein was more densely expressed in the nucleus than in the cytosol in transfected COS-7 cells. m-Neugrin-GFP protein also accumulated predominantly in nucleus. Control transfections with the empty vector showed that GFP was distributed throughout the entire cell (Fig. 5). FIG. 2. Amino-acid sequence comparison of Neugrin and m-Neugrin. Residues identical to each other and conservative substitutions are shaded in black and gray, respectively. Alignment gaps in the sequences are represented by “-.” An NLS-like domain is boxed.
Expression in Differentiated and Blastomal Cells SH-SY5Y cells, human neuroblastoma cells, were differentiated with RA treatment. Some SH-SY5Y cells
FIG. 3. Tissue distribution of neugrin and m-neugrin mRNA expression. (A) Human tissues, (B) human brain parts, (C) mouse tissues, and (D) mouse embryo Multiple Tissue Northern blots (Clontech) were hybridized with a probe of neugrin (A and B) or m-neugrin (C and D), labeled by Alk Phos Direct (Amersham Pharmacia). 529
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FIG. 4. In situ hybridization (ISH) analysis in human and mouse spinal cords. The transverse section of a human postmortem spinal cord was hybridized with digoxigenin-labeled cRNA probes of neugrin. The transverse section of an 11-week-old mouse spinal cord was used for ISH with m-neugrin probes. Signals were identified when using antisense but not sense probes.
began to extend neurites at 12 h treatment, and most of the cells differentiated to apparent neuronal phenotypes with long neurite processes by 72 h of treatment. Neuro2a cells, mouse neuroblastoma cells, were differentiated with RA and MG132, a proteasome inhibitor. In the presence of RA, cells began to extend bipolar neurites, and MG132 treatment also caused the cells to begin differentiating into spindle-like bipolar forms in 12 h. After 48 h of treatment, morphological differentiation seemed to be complete (Fig. 6A). A semi-quantitative RT-PCR showed that the RA treatment of SH-SY5Y cells upregulated the signals of the neugrin transcripts at 12 h and continued at a plateau level up to 72 h. The m-neugrin signals were also upregulated by the RA or MG132 treatment in neuro2a cells with time in culture (Fig. 6B). Northern blot analysis confirmed the RA- or MG132treated upregulation of the neugrin and m-neugrin ex-
pression in SH-SY5Y and neuro2a cells (SH-SY5Y: 72 h treatment of RA, neuro2a: 48 h treatment of RA or MG132). The RA- and MG132-treated upregulation of neugrin and m-neugrin seemed to parallel with the degree of morphological differentiation (Figs. 6A and 6B). DISCUSSION The neugrin and m-neugrin mRNA encode 219 and 230 amino acids, respectively, and do not have any distinctive motifs. Neugrin includes the NLS-like domain “RRRK;” however, this domain was “QKKR” in m-Neugrin. The subcellular localization of the Neugrin- and m-Neugrin-GFP proteins was in the nucleus despite the difference in amino acid sequence between “RRRK” in Neugrin and “QKKR” in m-Neugrin. The neugrin and m-neugrin were highly induced in association with neuronal differentiation in neuroblastoma
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FIG. 5. Subcellular localization of Neugrin-GFP and m-Neugrin-GFP protein. COS-7 cells were transiently transfected with GFP fusion constructs (Neugrin-GFP and m-Neugrin-GFP) or empty vector (Mock). Recombinant fusion proteins were visualized by direct fluorescence microscopy. The nucleus was stained by Hoechst 33342.
cells. Since those two genes have a 70% identity in amino acid sequence and similar subcellular localization, as well as similar expression profiles in neuronal differentiation, their evolutional process and functional roles are probably closely related. However, the distribution of neugrin in human tissues and m-neugrin in mouse tissues was somewhat different. RA has profound effects in regulating a broad range of biological process including cell differentiation. Some genes like NF-B or Bcl-2 have been directly implicated in neuronal differentiation after RA treatment (1, 2, 15, 16). RA also upregulates integrin ␣ 1 and  1 during differentiation of neuroblastoma cells including the SH-SY5Y cell line, and contributes to the laminin-induced enhancement of neurite outgrowth (17, 18). RA may work downstream of the NGF signal pathways in mouse sensory neurons (19). Despite these findings, the precise molecular mechanism of RA pro-
moted differentiation still remains unclear. It is suggested that proteasome inhibitors induce cell differentiation through cell cycle arrest. Those agents have been found to cause cell cycle arrest in both the G 0/G 1 and G 2/M phases (7, 8). The arrest at G 1 phase is rather important for cell differentiation following the arrest of cell cycles (20). MG132 has been shown to inhibit ubiquitin-dependent proteolysis associated with an increase in ubiquitin-protein conjugates (10). Thus, some molecules in ubiquitin-dependent proteolysis could be involved in neuronal differentiation caused by proteasome inhibitors, but the candidate molecules have not been identified. RA and MG132, therefore, involve different pathways to differentiate blast cells and promote neurites. In this study, we showed that neugrin and its mouse homologue, m-neugrin, were upregulated with neuronal differentiation induced by either RA or MG132.
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FIG. 6. Effects of differentiation-promoting agents in SH-SY5Y and neuro2a cells. (A) morphological change with time. (a) SH-SY5Y cells were treated with retinoic acid (RA) for the indicated time intervals. (b) Neuro2a cells with RA treatment. (c) Neuro2a cells with MG132 treatment. (B) Expression comparison between differentiated and blastoma cells. (a) Expression of neugrin in SH-SY5Y cells with or without RA. (b) Expression of m-neugrin in neuro2a cells with or without RA or MG 132. Semi-quantitative RT-PCR for the indicated time interval of RA or MG132 treatments is shown at the left. GAP-43 was used as a marker of neuronal differentiation, and -actin as an internal standard marker. Northern blotting hybridization with 2 g poly (A ⫹) RNA of SH-SY5Y and neuro2a cells treated with or without RA or MG132 is shown at the right. -actin was used as an internal control.
The expression of both genes nearly paralleled the degree of neural outgrowth and the expression of GAP43, a marker of neuronal differentiation. It is difficult to distinguish whether these genes are essential to
inducing the cells to differentiate or inducing the differentiation-related markers of cells. However, the fact that both RA and MG132 commonly caused upregulation of m-neugrin in neuro2a cells suggests that
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this gene was involved in both differentiation pathways induced by both RA and MG132. In other words, neugrin/m-neugrin may be common molecules directly associated with neuronal differentiation. The increasing level of mRNA with age in mouse embryonal tissues and its high expression in neurons in the neuronal tissues may support this view. These new genes may conceivably play an important role in neuronal cell differentiation or the maintenance of neuronal function in the nervous system. Further investigation and more evidence are needed to elucidate their precise mechanism in neurons, including anterior motor neurons, for which very few specific molecules have been identified.
13. 14.
REFERENCES
15.
1. Feng, Z., and Porter, A. G. (1999) J. Biochem. 274, 30341–30344. 2. Lasorella, A., Iavarona, A., and Israel, M. A. (1995) Cancer Res. 55, 4711– 4716. 3. Paglini, G., Pigino, G., Kunda, P., Morfini, G., Maccioni, R., Quiroga, S., Ferreira, A., and Ca´ceres, A. (1998) J. Neurosci. 18, 9858 –9869. 4. Liu, H., Nakagawa, T., Kanematsu, T., Uchida, T., and Tsuji, S. (1999) J. Neurochem. 72, 1781–1790. 5. Prinetti, A., Bassi, R., Riboni, L., and Tettamanti, G. (1997) FEBS Lett. 414, 475– 479. 6. Hasagawa, T., Yamaguchi, K., Wada, T., Takeda, A., Itoyama, Y., and Miyagi, T. (2000) J. Biochem. 275, 8007– 8015. 7. Fenteany, G., Standaert, R. F., Reichard, G. A., Corey, E. J., and
8. 9.
10. 11. 12.
16. 17. 18.
19. 20.
533
Schreiber, S. L. (1994) Proc. Natl. Acad. Sci. USA 91, 3358 – 3362. Fenteany, G., and Schreiber, S. L. (1998) J. Biochem. 273, 8545– 8548. Encinas, M., Iglesias, M., Liu, Y., Wang, H., Muhaisen, A., Cen˜a, V., Gallego, C., and Comella, J. X. (2000) J. Neurochem. 75, 991–1003. Obin, M., Mesco, E, Gong, X., Haas, A. L., Joseph, J., and Taylor, A. (1999) J. Biochem. 274, 11789 –11795. Tsuji, S., Yamashita, T., Tanaka, M., and Nagai, Y. (1988) J. Neurochem. 50, 414 – 423. Mitsui, K., Tsuji, S., Yamazaki, M., and Nagai, Y. (1991) J. Neurochem. 57, 556 –561. Kato, K. (1995) Nuclear Acids Res. 23, 3685–3690. Doyu, M., Sawada, K., Mitsuma, N., Niwa, J., Yoshimoto, M., Fujii, Y., Sobue, G., and Kato, K. Mol. Brain Res. in press. Hanada, M., Krajewski, S., Tanaka, S., Cazals-Hatem, D., Spengler, B. A., Ross, R. A., Biedler, J. L., and Reed, J. C. (1993) Cancer Res. 53, 4978 – 4986. Katoh, S., Mitsui, Y., Kitani, K., and Suzuki, T. (1996) Biochemi. Biophys. Res. Commun. 229, 653– 657. Li, B.-S., Zhang, L., Gu, J., Amin, N. D., and Pant, H. C. (2000) J. Neurosci. 20, 6055– 6062. Rossino, P., Gavazzi, I., Timpl, R., Aumailley, M., Abbadini, M., Giancotti, F., Silengo, L., Marchisio, P. C., and Tarone, G. (1990) Exp. Cell Res. 189, 100 –108. Corcoran, J., and Maden, M. (1999) Nature Neurosci. 2, 307–308. Inokoshi, J., Katagiri, M., Arima, S., Tanaka, H., Hayashi, M., Kim, Y. B., Furumai, R., Yoshida, M., Horinouchi, S., and Omura, S. (1999) Biochemi. Biophys. Res. Commun. 256, 372– 376.
Two Novel Genes, Human neugrin and Mouse m-neugrin, Are Upregulated with Neuronal Differentiation in Neuroblastoma Cells Shinsuke Ishigaki, Jun-ichi Niwa, Tsuyoshi Yoshihara, Norimasa Mitsuma, Manabu Doyu, and Gen Sobue 1 Department of Neurology, Nagoya University, School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, Aichi 466-8550, Japan
Received November 8, 2000
We herein report two new genes, human neugrin and mouse homologue m-neugrin, found by screening the cDNA library for the human spinal cord. The neugrin mRNA encodes 219 amino acids and its deduced amino acid sequence contains an NLS-like domain. No previously known motif is found in it. m-neugrin mRNA encodes 233 amino acids. Neugrin and m-Neugrin are 70% homologous in amino acid sequence. Northern analysis revealed that neugrin was strongly expressed in the heart, brain, and skeletal muscle, and m-neugrin in the liver, kidney, and brain. A transfection study indicated that these proteins are localized in the nucleus. Although the expression of neugrin was found to be ubiquitous in the nervous system, in situ hybridization showed that both neugrin and m-neugrin were expressed mainly in the neurons rather than the glial cells. Their expression was highly upregulated with the neurite outgrowth associated with neuronal differentiation in neuroblastoma cell lines. These results indicate that neugrin and m-neugrin are mainly expressed in neurons in the nervous system, and play an important role in the process of neuronal differentiation. © 2000 Academic Press
A wide variety of gene expression has been reported in association with neuronal differentiation (1– 6), and numerous stimuli, including that from retinoic acid (RA) and proteasome inhibitors, are known to cause neuronal differentiation with neurite outgrowth in neuronal cell lines (7–10). In terms of morphology, various factors act to promote neuronal differentiation, and it follows that these different factors promote neuronal differentiation on the basis of different molecular mechanisms (7, 8, 11, 12). In this report, human neuTo whom correspondence should be addressed. Fax: ⫹81-52-7442384. E-mail: [email protected]. 1
0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
grin (neurite outgrowth associated protein) and its mouse homologue, m-neugrin, new genes which are upregulated in differentiated neural cells, are characterized in terms of tissue distribution, subcellular localization, and expression profile in differentiated neuronal cells and blastomal cells. MATERIALS AND METHODS Human spinal cord cDNA library. Spinal cord was obtained at autopsy. Autopsy was performed within 6 h post mortem. Total RNA samples prepared from the anterior horn specimens were reverse transcribed by MMLV reverse transcriptase (Superscript II, Gibco BRL). Then, cDNAs were cut with BsmFI and ligated to those from a pool of 64 biotinylated adaptors cohesive to all possible overhangs. Ligated molecules were digested by BsmAI and FokI, and were recovered with streptavidin-coated paramagnetic beads. PCR was performed with the adaptor-primer and an anchored oligo-dT primer. The amplified fragments were separated by polyacrilamide gel electrophoresis. By repeating the experiment with 64 adapters, three enzymes and three anchored oligo-dT primers, 254 profiles of cDNAs were established. This procedure has been described in detail in previous reports (13, 14). Molecular cloning of neugrin. One of the cDNAs in the library described above was named neugrin, which was obtained with the 58th adapter, digested by BsmFI, and amplified by G-based anchored oligo-dT primers. The full length of the cDNA was obtained with the use of appropriate PCR based cloning methods including RACE PCR. Identification of m-neugrin. A search for murine expressed sequence tags (ESTs) identical to the neugrin fragment was made on the EST database at the National Center for Biotechnology Information (NCBI) using the BLASTN program. PCR with primers that are identical to both neugrin and the BLASTN hit EST, mouse EST No. AA815517, led to a fragment of the mouse homologue of neugrin. The full-length sequence of the gene was then determined by 5⬘ and 3⬘ RACE methods. Northern blot analysis. A sample of poly (A ⫹) RNA (2 g) was isolated from neuro2a cells or SH-SY5Y cells with respective agents (Micro-Fast Track 2.0, Invitrogen), then separated by electrophoresis through denaturing in 1% formaldehyde and transferred on to nylon membranes (Hybond N ⫹, Amersham Pharmacia Biotech). Human and mouse Multiple Tissue Northern blots were purchased from Clontech. Membranes were hybridized with a probe of neugrin, m-neugrin, or -actin, which was labeled by Alk Phos Direct (Amer-
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sham Pharmacia). The probes were obtained by denaturing the cDNA corresponding to either neugrin or m-neugrin (neugrin: 452 bp, 1307 to 1758, m-neugrin: 693 bp, 261 to 953). The -actin probe was purchased from Clontech. After overnight incubation at 55°C, membranes were washed with primary wash buffer and secondary buffer as described in the manufacturer’s specifications. The signals were then chemiluminiscently detected with CDP star (Amersham Pharmacia). The membranes were initially hybridized with the probes encoding neugrin or m-neugrin. Hybridization with the -actin probe followed as an internal control. In situ hybridization (ISH). Frozen sections (6 m thick) were prepared and fixed in 4% paraformaldehyde immediately. The deparaffinized sections were treated with proteinase K, refixed in 4% paraformaldehyde, and were prepared for in situ hybridization. Digoxigenin-labeled cRNA probes of 450 bp were generated for in situ hybridization from linearized plasmids for neugrin and m-neugrin, using Sp6 or T7 polymerase (Boehringer Mannheim). Control sense probes were also generated from each plasmid using the reciprocal RNA polymerase. Sliced tissues were hybridized for 16 h at 40°C with these cRNA probes in solution (50% formaldehyde, 10 mM Tris–HCl, pH 7.6, 1 mM EDTA, pH 8.0, 0.6 M NaCl, 0.25% SDS, 0.1 mg/ml yeast tRNA, 10% dextran sulfate, and 1 ⫻ Denhardt’s solution). The samples were then washed at room temperature in formamide/SSC (1:1) followed by 30 min at 50°C in 1 ⫻ SSC. The signals were detected immunologically with alkaline phosphatase-conjected anti-digoxigenin antibody according to the manufacturer’s protocol (Boehringer Mannheim). Expression of green fluorescent protein (GFP) fusion proteins and subcellular localization in COS-7 cells. To construct expression clones for Neugrin-GFP and m-Neugrin-GFP fusion proteins, the coding regions of neugrin and m-neugrin were amplified from human and mouse brain cDNA libraries. Restriction sites (XhoI and BamHI) for cloning into pEGFP-N1 (Clontech) were introduced by appropriate design of the PCR primers. A pEGFP-C3 (Clontech) vector without any constructs was used as a blank vector. The cloned PCR product was sequenced absolutely to exclude PCR-generated errors. Half g of each vector was preincubated with 5 l of Effectine reagent (Quiagen) diluted in 350 l medium. Transfection was performed according to the manufacturer’s protocol. The subcellular localization of GFP fusion protein in transiently transfected COS-7 cells was evaluated by microscopic study. Cell culture and differentiation. Cells were cultured in DMEM with 10% fetal calf serum (FCS) and 1% penicillin sulfate. Cells were maintained at 37°C in a saturated humidity atmosphere containing 95% air and 5% CO 2, and were seeded at an initial density of 10 4 cells per cm 2 in dishes. SH-SY5Y cells were treated in DMEM with 5% FCS and 10 M RA for 12, 24, 48, and 72 h for differentiation. Differentiation of neuro2a cells was induced in DMEM with 2% FCS and 5 M RA or DMEM with 10% FCS and 0.5 M N-carbobenzoxylLeu-Leu-leucinal (MG132), a proteasome inhibitor. Cells were harvested after incubation in medium with each agent for 12, 24, 48 and 72 h. Control cells were harvested without agents. RT-PCR analysis. Total RNA (2 g) was isolated from differentiated 2 ⫻ 10 5 neuro2a cells and SH-SY5Y cells using Micro RNA Isolation Kit (Strategene). cDNAs were synthesized by Super Script II. Then, cDNAs were amplified with specific primers for neugrin and m-neugrin. GAP-43 primers were used as a marker of neuronal differentiation. -actin primers were used as an internal control. The PCR primers used were 5⬘-GGGCATGAAGCCTCATGATCTAA-3⬘ (sense) and 5⬘-TATCCACGCAGCTCTTTCCT-3⬘ (antisense) for neugrin; 5⬘-CATGGAGCAGATCCGGTATT-3⬘ (sense) and 5⬘-TCTGAAGCTCTCGCTGATGA-3⬘ (antisense) for m-neugrin; 5⬘-AGAGCAGCCAAGCTGAAGAG-3⬘ (sense) and 5⬘-GAGGAAAGTGGACTCCCACA-3⬘ (antisense) for human GAP-43; 5⬘-CTAAGGAAAGTGCCCGACAG-3⬘; (sense) and 5⬘-TCGCCATAACAACACCAAGA-3⬘ (antisense) for mouse GAP-43; and 5⬘-CGTGCGTGACATTAAGGAGA-3⬘ (sense) and 5⬘-ACTCCTGCTTGCTGATCCAC-3⬘
(antisense) for human and mouse -actin. Each cycle consisted of denaturation at 94°C for 40 s, annealing at 60°C for 40 s, and extension at 72°C for 1 min. Appropriate numbers of PCR cycles for specific cDNAs were determined on the linear range of cycles, and also the linearity of the RT-PCR assays was ascertained with respect to the amounts of cDNA products. Aliquots of the PCR products were separated by 1% agarose gel electrophoresis and visualized by UVillumination after ethidium bromide staining.
RESULTS cDNA Cloning of neugrin and m-neugrin An 80 bp cDNA fragment was picked up from cDNA fragment profiles generated by BsmFI. The PCR primers, designed on the basis of the EST (human EST No. AA165380) that is identical for this fragment and RACE methods, yielded a specific product of 1780 bp, including a 660 bp open reading frame (ORF) (Fig. 1A, GenBank/EBI No. AB029315). A BLAST search revealed that one sequence of human DNA clone contained the entire neugrin gene (Gen Bank/EBI No. AL035467), and this clone has been mapped to 6q 12–13. A BLAST search using neugrin identified the highly homologous mouse EST (No. AA815517), and the full length of the neugrin mouse homologue of 1346 bp, including a deduced 693 bp ORF, named m-neugrin, was obtained using RACE methods (Fig. 1B). The sequence has been deposited in GenBank/EBI Data Bank under No. AB 047544. Structural Analysis The amino acid sequence alignment of both proteins shows an identity of 70%. There is an NLS-like domain from the 133rd to 136th, “RRRK” in the neugrin protein, Neugrin, although this domain changes into “QKKR” in m-Neugrin. No apparent previously known motif was found in either protein (Fig. 2). Tissue Distribution of neugrin and m-neugrin and Their Expression in the Nervous System In neugrin, Northern analysis of human MTN (Clontech) yielded a strong signal with RNA from the brain, heart, and skeletal muscle (Fig. 3A), while Northern analysis with m-neugrin exhibited relatively strong signals in the liver, kidney, heart, and brain (Fig. 3B). The pattern of neugrin mRNA expression in the human nervous system was rather ubiquitous (Fig. 3C). In human MTN, a shorter splice variant of neugrin was seen at 1.2 kbp. However, its expression level in the brain was not strong, and we investigated only the major molecule of neugrin in this study. Using mouse embryo MTN (Clontech), the expression pattern of the m-neugrin transcript in mouse embryo was examined. The level of m-neugrin mRNA increased with embryonal development (Fig. 3D).
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FIG. 1. Nucleotide and predicted amino acid sequences of neugrin (A) and m-neugrin (B). The complete sequence of neugrin (A) and m-neugrin (B) cDNA and their translation products are illustrated. The predicted amino acid sequence is shown from the first ATG codon of the open reading frame. Nucleotide and amino acid numbers are shown at the right. The GenBank Acc. No. for neugrin is AB029315. The GenBank Acc. No. for m-neugrin is AB047544.
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For further analysis of the distribution of neugrin and m-neugrin expression in the nervous system, in situ hybridization was performed using the samples of adult human postmortem spinal cords and adult mouse spinal cords. These mRNA were mainly expressed in the motor neurons, but very weak signals were also detected in the glial cells (Fig. 4). Subcellular Localization Neugrin-GFP fusion protein was more densely expressed in the nucleus than in the cytosol in transfected COS-7 cells. m-Neugrin-GFP protein also accumulated predominantly in nucleus. Control transfections with the empty vector showed that GFP was distributed throughout the entire cell (Fig. 5). FIG. 2. Amino-acid sequence comparison of Neugrin and m-Neugrin. Residues identical to each other and conservative substitutions are shaded in black and gray, respectively. Alignment gaps in the sequences are represented by “-.” An NLS-like domain is boxed.
Expression in Differentiated and Blastomal Cells SH-SY5Y cells, human neuroblastoma cells, were differentiated with RA treatment. Some SH-SY5Y cells
FIG. 3. Tissue distribution of neugrin and m-neugrin mRNA expression. (A) Human tissues, (B) human brain parts, (C) mouse tissues, and (D) mouse embryo Multiple Tissue Northern blots (Clontech) were hybridized with a probe of neugrin (A and B) or m-neugrin (C and D), labeled by Alk Phos Direct (Amersham Pharmacia). 529
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FIG. 4. In situ hybridization (ISH) analysis in human and mouse spinal cords. The transverse section of a human postmortem spinal cord was hybridized with digoxigenin-labeled cRNA probes of neugrin. The transverse section of an 11-week-old mouse spinal cord was used for ISH with m-neugrin probes. Signals were identified when using antisense but not sense probes.
began to extend neurites at 12 h treatment, and most of the cells differentiated to apparent neuronal phenotypes with long neurite processes by 72 h of treatment. Neuro2a cells, mouse neuroblastoma cells, were differentiated with RA and MG132, a proteasome inhibitor. In the presence of RA, cells began to extend bipolar neurites, and MG132 treatment also caused the cells to begin differentiating into spindle-like bipolar forms in 12 h. After 48 h of treatment, morphological differentiation seemed to be complete (Fig. 6A). A semi-quantitative RT-PCR showed that the RA treatment of SH-SY5Y cells upregulated the signals of the neugrin transcripts at 12 h and continued at a plateau level up to 72 h. The m-neugrin signals were also upregulated by the RA or MG132 treatment in neuro2a cells with time in culture (Fig. 6B). Northern blot analysis confirmed the RA- or MG132treated upregulation of the neugrin and m-neugrin ex-
pression in SH-SY5Y and neuro2a cells (SH-SY5Y: 72 h treatment of RA, neuro2a: 48 h treatment of RA or MG132). The RA- and MG132-treated upregulation of neugrin and m-neugrin seemed to parallel with the degree of morphological differentiation (Figs. 6A and 6B). DISCUSSION The neugrin and m-neugrin mRNA encode 219 and 230 amino acids, respectively, and do not have any distinctive motifs. Neugrin includes the NLS-like domain “RRRK;” however, this domain was “QKKR” in m-Neugrin. The subcellular localization of the Neugrin- and m-Neugrin-GFP proteins was in the nucleus despite the difference in amino acid sequence between “RRRK” in Neugrin and “QKKR” in m-Neugrin. The neugrin and m-neugrin were highly induced in association with neuronal differentiation in neuroblastoma
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FIG. 5. Subcellular localization of Neugrin-GFP and m-Neugrin-GFP protein. COS-7 cells were transiently transfected with GFP fusion constructs (Neugrin-GFP and m-Neugrin-GFP) or empty vector (Mock). Recombinant fusion proteins were visualized by direct fluorescence microscopy. The nucleus was stained by Hoechst 33342.
cells. Since those two genes have a 70% identity in amino acid sequence and similar subcellular localization, as well as similar expression profiles in neuronal differentiation, their evolutional process and functional roles are probably closely related. However, the distribution of neugrin in human tissues and m-neugrin in mouse tissues was somewhat different. RA has profound effects in regulating a broad range of biological process including cell differentiation. Some genes like NF-B or Bcl-2 have been directly implicated in neuronal differentiation after RA treatment (1, 2, 15, 16). RA also upregulates integrin ␣ 1 and  1 during differentiation of neuroblastoma cells including the SH-SY5Y cell line, and contributes to the laminin-induced enhancement of neurite outgrowth (17, 18). RA may work downstream of the NGF signal pathways in mouse sensory neurons (19). Despite these findings, the precise molecular mechanism of RA pro-
moted differentiation still remains unclear. It is suggested that proteasome inhibitors induce cell differentiation through cell cycle arrest. Those agents have been found to cause cell cycle arrest in both the G 0/G 1 and G 2/M phases (7, 8). The arrest at G 1 phase is rather important for cell differentiation following the arrest of cell cycles (20). MG132 has been shown to inhibit ubiquitin-dependent proteolysis associated with an increase in ubiquitin-protein conjugates (10). Thus, some molecules in ubiquitin-dependent proteolysis could be involved in neuronal differentiation caused by proteasome inhibitors, but the candidate molecules have not been identified. RA and MG132, therefore, involve different pathways to differentiate blast cells and promote neurites. In this study, we showed that neugrin and its mouse homologue, m-neugrin, were upregulated with neuronal differentiation induced by either RA or MG132.
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FIG. 6. Effects of differentiation-promoting agents in SH-SY5Y and neuro2a cells. (A) morphological change with time. (a) SH-SY5Y cells were treated with retinoic acid (RA) for the indicated time intervals. (b) Neuro2a cells with RA treatment. (c) Neuro2a cells with MG132 treatment. (B) Expression comparison between differentiated and blastoma cells. (a) Expression of neugrin in SH-SY5Y cells with or without RA. (b) Expression of m-neugrin in neuro2a cells with or without RA or MG 132. Semi-quantitative RT-PCR for the indicated time interval of RA or MG132 treatments is shown at the left. GAP-43 was used as a marker of neuronal differentiation, and -actin as an internal standard marker. Northern blotting hybridization with 2 g poly (A ⫹) RNA of SH-SY5Y and neuro2a cells treated with or without RA or MG132 is shown at the right. -actin was used as an internal control.
The expression of both genes nearly paralleled the degree of neural outgrowth and the expression of GAP43, a marker of neuronal differentiation. It is difficult to distinguish whether these genes are essential to
inducing the cells to differentiate or inducing the differentiation-related markers of cells. However, the fact that both RA and MG132 commonly caused upregulation of m-neugrin in neuro2a cells suggests that
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this gene was involved in both differentiation pathways induced by both RA and MG132. In other words, neugrin/m-neugrin may be common molecules directly associated with neuronal differentiation. The increasing level of mRNA with age in mouse embryonal tissues and its high expression in neurons in the neuronal tissues may support this view. These new genes may conceivably play an important role in neuronal cell differentiation or the maintenance of neuronal function in the nervous system. Further investigation and more evidence are needed to elucidate their precise mechanism in neurons, including anterior motor neurons, for which very few specific molecules have been identified.
13. 14.
REFERENCES
15.
1. Feng, Z., and Porter, A. G. (1999) J. Biochem. 274, 30341–30344. 2. Lasorella, A., Iavarona, A., and Israel, M. A. (1995) Cancer Res. 55, 4711– 4716. 3. Paglini, G., Pigino, G., Kunda, P., Morfini, G., Maccioni, R., Quiroga, S., Ferreira, A., and Ca´ceres, A. (1998) J. Neurosci. 18, 9858 –9869. 4. Liu, H., Nakagawa, T., Kanematsu, T., Uchida, T., and Tsuji, S. (1999) J. Neurochem. 72, 1781–1790. 5. Prinetti, A., Bassi, R., Riboni, L., and Tettamanti, G. (1997) FEBS Lett. 414, 475– 479. 6. Hasagawa, T., Yamaguchi, K., Wada, T., Takeda, A., Itoyama, Y., and Miyagi, T. (2000) J. Biochem. 275, 8007– 8015. 7. Fenteany, G., Standaert, R. F., Reichard, G. A., Corey, E. J., and
8. 9.
10. 11. 12.
16. 17. 18.
19. 20.
533
Schreiber, S. L. (1994) Proc. Natl. Acad. Sci. USA 91, 3358 – 3362. Fenteany, G., and Schreiber, S. L. (1998) J. Biochem. 273, 8545– 8548. Encinas, M., Iglesias, M., Liu, Y., Wang, H., Muhaisen, A., Cen˜a, V., Gallego, C., and Comella, J. X. (2000) J. Neurochem. 75, 991–1003. Obin, M., Mesco, E, Gong, X., Haas, A. L., Joseph, J., and Taylor, A. (1999) J. Biochem. 274, 11789 –11795. Tsuji, S., Yamashita, T., Tanaka, M., and Nagai, Y. (1988) J. Neurochem. 50, 414 – 423. Mitsui, K., Tsuji, S., Yamazaki, M., and Nagai, Y. (1991) J. Neurochem. 57, 556 –561. Kato, K. (1995) Nuclear Acids Res. 23, 3685–3690. Doyu, M., Sawada, K., Mitsuma, N., Niwa, J., Yoshimoto, M., Fujii, Y., Sobue, G., and Kato, K. Mol. Brain Res. in press. Hanada, M., Krajewski, S., Tanaka, S., Cazals-Hatem, D., Spengler, B. A., Ross, R. A., Biedler, J. L., and Reed, J. C. (1993) Cancer Res. 53, 4978 – 4986. Katoh, S., Mitsui, Y., Kitani, K., and Suzuki, T. (1996) Biochemi. Biophys. Res. Commun. 229, 653– 657. Li, B.-S., Zhang, L., Gu, J., Amin, N. D., and Pant, H. C. (2000) J. Neurosci. 20, 6055– 6062. Rossino, P., Gavazzi, I., Timpl, R., Aumailley, M., Abbadini, M., Giancotti, F., Silengo, L., Marchisio, P. C., and Tarone, G. (1990) Exp. Cell Res. 189, 100 –108. Corcoran, J., and Maden, M. (1999) Nature Neurosci. 2, 307–308. Inokoshi, J., Katagiri, M., Arima, S., Tanaka, H., Hayashi, M., Kim, Y. B., Furumai, R., Yoshida, M., Horinouchi, S., and Omura, S. (1999) Biochemi. Biophys. Res. Commun. 256, 372– 376.