HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 281, Issue 11, 7498-7514, March 17, 2006

This Article Abstract Full Text (PDF) All Versions of this Article: 281/11/7498    most recent M510881200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Urano, Y. Articles by Tashiro, F. Search for Related Content PubMed PubMed Citation Articles by Urano, Y. Articles by Tashiro, F. Social Bookmarking                      What's this?

Involvement of the Mouse Prp19 Gene in Neuronal/Astroglial Cell Fate Decisions^*

From the Department of Biological Science and Technology, Faculty of Industrial Science and Technology, Tokyo University of Science, Yamazaki, Noda-shi, Chiba 270-8510, Japan

Received for publication, October 5, 2005 , and in revised form, December 12, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The molecular mechanisms involved in neuronal/astroglial cell fate decisions during the development of the mammalian central nervous system are poorly understood. Here, we report that PRP19, a splice variant of mouse PRP19 corresponding to the yeast PRP19 protein, can function as a neuron-astroglial switch during the retinoic acid-primed neural differentiation of P19 cells. The -variant possesses an additional 19 amino acid residues inframe in the N-terminal region of the -variant. The forced expression of the -variant RNA caused the down-regulation of oct-3/4 and nanog mRNA expression during the 12-48 h of the late-early stages of neural differentiation and was sufficient to convert P19 cells into neurons (but not glial cells) when the cells were cultured in aggregated form without retinoic acid. In contrast, the forced expression of the -variant RNA suppressed neuronal differentiation and conversely stimulated astroglial cell differentiation in retinoic acid-primed P19 cells. Based on yeast two-hybrid screening, cyclophilin A was identified as a specific binding partner of the -variant. Luciferase reporter assay mediated by the oct-3/4 promoter revealed that cyclophilin A could act as a transcriptional activator and that its activity was suppressed by the -variant, suggesting that cyclophilin A takes part in the induction of oct-3/4 gene expression, which might lead to neuroectodermal otx2 expression within 12 h of the immediate-early stages of retinoic acid-primed neural differentiation. These results show that the -variant gene plays a pivotal role in neural differentiation and that the -variant participates in neuronal/astroglial cell fate decisions.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In mammals, the neural tube consists of a morphologically identical population of neuroepithelial cells from which all the diverse neuronal and glial subtypes of the central nervous system are generated. As neurogenesis begins, proliferating neural progenitors become restricted to the ventricular zone at the inner surface of the neural tube. Stem cells within the ventricular zone cease to produce neurons at around midgestation and instead start to produce glia. What causes these stem cells to switch from neurogenesis to gliogenesis is one of the most important issues in neural differentiation.

In Drosophila, neuronal and glial fates are determined by the gcm (glial cell missing) gene encoding a transcription factor as a binary switch (1). In a gcm mutant, glial cells are converted to neurons, whereas ectopic expression of gcm in neurons causes neuron-glial transformation. Two mammalian gcm homologs, Gcm1 and Gcm2, have been identified in mice, rats, and humans (2-6); however, the sites of Gcm1 and Gcm2 expression are largely restricted to the placenta (6, 7) and para-thyroid glands (6), respectively. Targeted disruption of the Gcm1 gene in mice results in a severe defect in labyrinth formation in the placenta (8, 9). Introduction of the Gcm1 gene into cultured embryonic brain cells results in the induction of an astrocyte lineage. Nonetheless, cultures from Gcm1-deficient mouse brain do not exhibit a significant reduction in the number of astrocytes, suggesting that mouse Gcm1 exhibits the potential to induce gliogenesis, but may function in the generation of a minor subpopulation of glial cells (10). Furthermore, Gcm2-targeted mice exhibit a selective loss of parathyroid glands, but no abnormalities were reported in the nervous system (11). Thus, in mammals, although there is evidence that neurons and glia share a common progenitor (12), the genes that might control a GCM-like switching mechanism have not yet been identified.

Recent studies have shown that the Sox9 transcription factor (13), the QKI RNA-binding protein (14), and extrinsic cues such as bone morphogenetic protein (BMP)² and ciliary neurotrophic factor (CNTF) (15) promote the differentiation of neural progenitors into glia. Nevertheless, these limited factors may be insufficient for the regulation of glial cell differentiation because mammalian astrocytes exhibit a large heterogeneity, differing in morphology, distribution, molecular types expressed, function, and cell lineage.

In this context, to clarify the molecular mechanisms in neuronal/astroglial fate decisions in mammals, we applied a subtractive cDNA cloning strategy for the retinoic acid (RA)-induced neural differentiation system of mouse P19 embryonic carcinoma cells as described previously (16). By this approach, we isolated PRP19 (precursor RNA processing-19) and its splice variant PRP19, with an additional 19-amino acid residues in-frame. PRP19 and PRP19 are homologs of yeast Prp19, which has been identified in a screen of temperature-sensitive mutants defective in precursor RNA processing of Saccharomyces cerevisiae and shown to be essential for pre-mRNA splicing (17). Nonetheless, there is no report concerning the participation of PRP19 and PRP19 in neural differentiation so far.

In this study, we show that P19 cells modified to express PRP19 constitutively in large quantities no longer required RA for neuronal differentiation. When these cells were cultured in aggregated form, the mRNA expression of neurogenic basic helix-loop-helix (bHLH) transcription factors, including Mash-1, Ngn-1 (neurogenin-1), and NeuroD, was promoted to sufficient levels for the induction of -tubulin III-positive neurons, but not glial fibrillary acidic protein (GFAP)-positive astroglia, accompanied by the down-regulation of oct-3/4 and nanog mRNA expression. On the other hand, the forced expression of Prp19 RNA suppressed neuronal differentiation through the inhibition of CypA (cyclophilin A), which acts as a transcriptional activator, and inversely promoted glial cell differentiation in RA-primed P19 cells. The down-regulation of Prp19 RNA by the antisense 15-mer oligodeoxynucleotides (ODNs) specific for the insertional sequence of Prp19 stimulated neuronal differentiation and suppressed astroglial cell differentiation in RA-primed P19 cells. These results imply that the regulation of alternative splicing of the mouse Prp19 gene plays an important role in neuronal and astroglial cell fate decisions.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cell Cultures and Experimental Animals—Mouse P19 embryonic carcinoma cells were obtained from American Type Culture Collection (Manassas, VA). To induce neural differentiation, 1 x 10⁶ aggregated P19 cells were cultured in 10-cm bacteriological grade dishes in 10 ml of -minimal essential medium containing 10% fetal calf serum and 5 x 10^-7 M all-trans-RA (Sigma) for 4 days as described previously (16). The cell aggregates were suspended by mild pipetting and transferred to tissue culture dishes. The cells were cultured in RA-free -minimal essential medium containing 10% fetal calf serum for an additional 4 days to induce -tubulin III-positive neurons and for 7 days to induce GFAP-positive astroglial cells. ICR mice were purchased from Charles River Japan (Kanagawa, Japan).

Culture of Mouse Neural Stem (NS) Cells—NS cells from hippocampi of ICR mice at postnatal day 7 were cultured in serum-free Dulbecco's modified Eagle's medium/F-12 nutrient mixture containing 20 ng/ml mouse epidermal growth factor (EGF; Sigma) as described previously (18). To induce neural differentiation, the spheres formed by NS cells were plated onto tissue culture dishes precoated with 20 mg/ml poly-L-lysine and cultured in EGF-free medium containing 20 ng/ml recombinant rat CNTF (R&D Systems, Minneapolis, MN) for 2 weeks.

Subtractive Cloning—Tester poly(A)⁺ RNA was prepared from aggregated P19 cells treated with 5 x 10^-7 M RA for 24 h, and firststrand cDNA was hybridized with excess driver RNA prepared from untreated P19 cells, and then a subtractive cDNA probe was prepared by the addition of the cross-linking agent 2,5-diaziridinyl-1,4-benzoquinone and used to screen 9 x 10⁵ plaques of rat brain 5'-stretch plus a gt11 cDNA library (Clontech) as described previously (16). The cDNAs inserted into the positive phage were then amplified by PCR using primers based on the phage DNA sequence: 5'-primer, 5'-GCC ACG ACT CCT GGA GCC CG-3'; and 3'-primer, 5'-CAC CAG ACC AAC TGG TAA TG-3'. The amplified cDNAs were digested with EcoRI and subcloned into the EcoRI site of pGEM-7zf(+) (Promega Corp., Madison, WI).

A rat full-length Prp19 cDNA clone was isolated from the second screening of 4.5 x 10⁵ plaques from a rat brain gt10 cDNA library (Clontech) using the 1.1-kb EcoRI cDNA fragment obtained from the first screening as a probe and then subcloned into the EcoRI site of pGEM-7zf(+). The plasmid was designated as pGEM-Prp19.

Primers Used for PCR Amplification—Total RNAs extracted from the spheres of P19 cells and mouse NS cells were reverse-transcribed, and cDNAs were amplified by PCR as described previously (18). The primer sequences used were as follows: mouse Prp19 and Prp19, 5'-CGC CAT GTC CCT GAT CTG CTC G-3' (5'-primer) and 5'-GAT GCC ACC TGC CGG TAC TTG C-3')(3'-primer); glyceraldehyde-3-phosphate dehydrogenase, 5'-CCA CAG TCC ATG CCA TCA CTG CC-3' (5'-primer) and 5'-GGC AGT GAT GGC ATG GAC TGT GG-3' (3'-primer) (19); ribosomal phosphoprotein, 5'-CAG CTC TGG AGA AAC TGC TG-3' (5'-primer) and 5'-GTG TAC TCA GTC TCC ACA GA-3' (3'-primer) (20); oct-3/4, 5'-CCT GGC TAA GCT TCC AAG GGC-3' (5'-primer) and 5'-GTT CTA GCT CCT TCT GCA GGG C-3' (3'-primer) (21); nanog,5'-CTA TGA TCT TTC CTT CTA GAC ACT G-3' (5'-primer) and 5'-AGC CCA AAG CTT GCG TAA GTC TCA TAT T-3' (3'-primer) (22, 23); Mash-1,5'-CAC AAG TCA GCG GCC AAG CAG-3' (5'-primer) and 5'-GAT CCC TCG TCG GAG GAG TAG-3' (3'-primer) (24); Ngn-1, 5'-CCT TTG GAG ACC TGC ATC TC-3' (5'-primer) and 5'-GAT GTA GTT GTA GGC GAA GC-3' (3'-primer) (25); NeuroD, 5'-GCA TGC ACG GGC TGA ACG C-3' (5'-primer) and 5'-GGG ATG CAC CGG GAA GGA AG-3' (3'-primer) (26); GFAP, 5'-TTT CTC CTT GTC TCG AAT GA-3' (5'-primer) and 5'-GGT TTC ATC TTG GAG CTT CT-3' (3'-primer) (25); otx2,5'-CCG ACT TTG CGC CTC CAA ACA A-3' (5'-primer) and 5'-GGT TGA TGG ACC CTT CTA AGG C-3' (3'-primer) (27); retinoic acid receptor (RAR), 5'-CAT CAC AAC TAC CTG CCA GAC TC-3' (5'-primer) and 5'-GTG CGC TTT GCG AAC CTT CTC AA-3' (3'-primer) (28); RAR, 5'-CGC CTC CAC ACC TAG AGG ATA AG-3' (5'-primer) and 5'-TGG GGA ATG TCT GCA ACA GCT GG-3' (3'-primer) (29); and CypA,5'-CGC GAA TTC CAG CCA TGG TCA ACC CCA C-3' (5'-primer) and 5'-CCA CTC GAG TTA GAG CTG TCC ACA GTC G-3' (3'-primer) (30).

The Prp19 PCR product was subcloned into the EcoRV site of pGEM-5zf(+) and designated as pGEM-Prp19. The CypA PCR product was digested with NsiI and inserted into the EcoRI-XhoI site of pACT2 (Clontech) and named pACT2-CypA.

Real-time PCR Analysis of PRP19 and PRP19 Expression during Neural Differentiation—To generate cDNA from P19 cell culture samples, 5 µg of total RNAs was reverse-transcribed with random primer. Real-time PCR was performed using SYBR Green PCR Master Mix (Eurogentec, Seraine, Belgium) according to the manufacturer's protocol. The specific primers used were as follows: Prp19, 5'-ATC AAC AAC CAG CCT CTC TCA GAG-3' (5'-primer) and 5'-TGC AGC ATG ACT GCA TCC C-3' (3'-primer); and Prp19, 5'-CAG AGG AGC AGC TCA TCG ACA-3' (5'-primer) and 5'-AGG GTA GGC CAC TGT GAG GAC T-3' (3'-primer). Fluorescence was detected at the end of each cycle to monitor the amount of PCR product. The quantities of specific mRNA in the sample were measured according to the corresponding gene-specific standard curve. Amplification of specific transcripts was confirmed by a final melting curve profile and gel electrophoresis. The levels of Prp19 and Prp19 transcripts at each time point were calibrated as compared with those of ribosomal phosphoprotein transcripts as an internal control (31).

Northern Blot Analysis—Aliquots of 20 µg of total RNAs were electrophoresed on 6% formaldehyde-containing 1% agarose gel, transferred to Hybond-N⁺ nylon membrane (Amersham Biosciences, Buckinghamshire, UK), and hybridized with ³²P-labeled PCR-amplified probe as described above. Developed x-ray films were scanned into a Macintosh G4, and the expression levels of these RNAs were quantified using the NIH Image 1.62 ppc program.

Construction of Expression Vectors—pcDNA3-EF1--sense Prp19 and pcDNA3-EF1--antisense Prp19 expression vectors were constructed by the insertion of the Prp19 cDNA fragment into the EcoRV site of pcDNA3-EF1- (16). The pcDNA3-EF1--sense Prp19 expression vector was constructed by the insertion of the ApaI-HindIII fragment of pGEM-Prp19 into the BamHI-HindIII site of pcDNA3-EF1--sense Prp19. pcDNA3-EF1--sense CypA and pcDNA3-EF1--antisense CypA expression vectors were constructed by the insertion of the blunt-ended EcoRI-XhoI CypA fragment of pACT2-CypA into the EcoRV site of pcDNA3-EF1-. These vectors were introduced into P19 cells by lipofection with N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium methyl sulfate (Roche Applied Science, Mannheim, Germany) and cultured in the presence of 400 µg/ml G418 (Wako, Tokyo, Japan) for selection. The stable transfectants of the pcDNA3-EF1--sense Prp19, pcDNA3-EF1--antisense Prp19, pcDNA3-EF1--sense Prp19, pcDNA3-EF1--sense CypA, and pcDNA3-EF1--antisense CypA vectors were designated as -S1, -A1, -S1, CypA-S1, and CypA-A1 cells, respectively.

View larger version (105K): [in this window] [in a new window]   FIGURE 1. Isolation of rat Prp19 cDNA by subtractive cloning. A, nucleotide and predicted amino acid sequences of rat Prp19 cDNA (DDBJ/GenBankTM/EBI accession number AB020022 [GenBank] ). B, amino acid sequence alignment of rat PRP19 protein with orthologs of various organisms: Rattus norvegicus (accession number NP_647549 [GenBank] ), M. musculus (accession number NP_598890 [GenBank] ), H. sapiens (accession number NP_055317 [GenBank] ), and S. cerevisiae (S.cere.; accession number P32523 [GenBank] ). The modified RING finger domain is boxed. The nuclear localization signal is underlined. Five putative WD40 repeats are shaded.

View larger version (77K): [in this window] [in a new window]   FIGURE 2. Expression of Prp19 mRNA, a splice variant of Prp19, during the neural differentiation of mouse hippocampus-derived NS cells in vitro. A, growth and differentiation of NS cells. NS cells from the mouse hippocampus at postnatal day 7 divided in a form of sphere in the presence of 20 ng/ml EGF (panel a). When these cells were cultured in the presence of 20 ng/ml CNTF for 2 weeks, they differentiated into both -tubulin III-positive neurons (panel b) and myelin basic protein (MBP)-positive oligodendrocytes (panel c). B, preferential induction of Prp19 mRNA during the CNTF-primed neural differentiation of NS cells. Total RNAs were extracted from untreated P19 cells and NS cells (NSCs) cultured with EGF or CNTF for 2 weeks and then analyzed by reverse transcription-PCR. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript was also analyzed by reverse transcription-PCR as an internal control. C, nucleotide and predicted amino acid sequences of mouse Prp19 cDNA. The putative ATG start site and termination codon are indicated at positions 5-7 and 1574-1576, respectively. The Prp19-specific sequence is highlighted in black. The modified RING finger domain, nuclear localization signal, and WD40 repeat are boxed, underlined, and shaded, respectively. The nucleotide sequence of mouse Prp19 cDNA was deposited in the DDBJ/GenBankTM/EBI Data Bank under accession number AB222602 [GenBank] .

Construction of Enhanced Green Fluorescent Protein (EGFP)-fused PRP19 and PRP19 Expression Vectors—EGFP-fused PRP19 and PRP19 expression vectors were constructed by insertion of the entire coding regions of PRP19 and PRP19, respectively, downstream of the EGFP-coding sequence in-frame into the pEGFP-C1 vector (Clontech) and designated as pEGFP-PRP19 and pEGFP-PRP19, respectively. P19 cells were transfected with the pEGFP-PRP19 and pEGFP-PRP19 expression vectors using Lipofectamine PLUS^TM (Invitrogen) and cultured in the presence of 400 µg/ml G418 for selection. To see the subcellular localization of PRP19 and PRP19, the cells were fixed with 4% paraformaldehyde and washed with Ca²⁺/Mg²⁺-free phosphate-buffered saline. The nuclei were stained with 1 µg/ml Hoechst 33258. The cells were observed under a fluorescence microscope (Axioplan 2, Carl Zeiss, Oberkochen, Germany).

View larger version (24K): [in this window] [in a new window]   FIGURE 3. Alternative splicing of Prp19 results in Prp19. A, exon-intron structure of the mouse Prp19 gene. The Prp19 gene was assigned to mouse chromosome 19 (DDBJ/GenBankTM/EBI accession number NT_039687 [GenBank] .1/Mm19_39727_35). Black boxes and horizontal lines indicate exons and introns, respectively. The Prp19-specific insertion sequence is presented as a gray box. B, nucleotide sequence of the intron 3-exon 4 junction of the mouse Prp19 gene. The Prp19-specific sequence is located just before exon 4 of the mouse Prp19 gene, and its predicted amino acid sequence is underlined. 3'ss, 3'-splice site. C, functional domains of PRP19 and PRP19 proteins. The PRP19 protein-specific insertion sequence encoding 19 amino acid residues in-frame in intron 3 of the Prp19 gene is presented as a hatched box. The modified RING finger domain, nuclear localization signal (NLS), and WD40 repeat are shown as white, black, and gray boxes, respectively. The total number of amino acid residues is indicated on the right of each protein. D, intracellular localization of PRP19 and PRP19 in P19 cells. P19 cells were transfected with EGFP-PRP19 (panel a) or EGFP-PRP19 (panel d). Nuclei were stained with Hoechst 33258 (panels b and e). At 48 h after transfection, the cells were observed under an Axioplan 2 fluorescence microscope. Panels c and f are merged images of panels a and b and panels d and e, respectively. Scale bar = 50 µm.

Immunoprecipitation—The hemagglutinin (HA)-fused CypA expression vector was constructed by insertion of the blunt-ended BglII open reading frame fragment of pACT2-CypA into the EcoRV site of pcDNA3-EF1-. P19 cells (3 x 10⁵) were transfected with 2 µg of DNA using Lipofectamine PLUS^TM. The cells were harvested 48 h after transfection and lysed with immunoprecipitation buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM MgCl₂, 0.1% Nonidet P-40, and 1 mM phenylmethylsulfonyl fluoride). For immunoprecipitations, equal amounts of cell lysate proteins were incubated overnight with protein A-Sepharose beads charged with anti-PRP19 or anti-HA antibody at 4 °C. The beads were washed twice with immunoprecipitation buffer. Immunoprecipitated proteins or cell lysates were mixed with 3x SDS sample buffer and separated by SDS-PAGE. Western blot analysis was performed as described above with the appropriate antibodies.

Gel Filtration of the Spliceosome—P19 cells (1 x 10⁸) were homogenized in lysis buffer (20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EGTA, 1 mM dithiothreitol, 10% glycerol, 0.1% Nonidet P-40, and 0.1 mM phenylmethylsulfonyl fluoride) and centrifuged at 105,000 x g for 1 h. The resulting supernatant was applied to a Sepharose 4B column (bed volume of 38.4 ml; Amersham Biosciences AB, Uppsala, Sweden) and eluted with 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EGTA, 1 mM dithiothreitol, 10% glycerol, and 0.1 mM phenylmethylsulfonyl fluoride. Fractions of 0.78 ml were collected and analyzed by Western blotting with the appropriate antibodies as described above.

Yeast Two-hybrid Screening—Mouse Prp19-specific region cDNA (amino acids 83-101) was inserted into pAS2-1 (Clontech) in-frame with the Gal4 DNA-binding domain as bait. An RA-treated P19 cell cDNA library was constructed using pACT2. Yeast two-hybrid screening was performed as described previously (34).

Chromatin Immunoprecipitation—P19 cells treated with RA for 0 and 6 h were cross-linked with 1% formaldehyde for 10 min. The nuclear fraction was isolated and sonicated to shear genomic chromatin; clarified by centrifugation; precleared with protein A-Sepharose; and immunoprecipitated with anti-CypA antibody, anti-RAR antibody (Santa Cruz Biotechnology, Inc.), or anti-acetylated histone H3 antibody (Upstate). Following immunoprecipitation and immobilization of immunocomplexes, proteinase K digestion was allowed to proceed for 1 h at 45 °C. DNA was purified by phenol/chloroform extraction. PCR was carried out on the purified DNA using primers to the oct-3/4 promoter region (RAREoct; -412 to +39, 5'-CTA GGA CTC GAG ACG GGT GGG TAA G-3' (5'-primer) and 5'-CAC CCG GAT CCG GGG GCC TGG-3' (3'-primer) (35)).

View larger version (32K): [in this window] [in a new window]   FIGURE 4. Induction of Prp19 and Prp19 gene expression during the RA-primed neural differentiation of P19 cells. Aggregated P19 cells were treated with 5 x 10-7 MRA in bacteriological grade dishes for 4 days. Cell aggregates were suspended with mild pipetting and replated onto tissue culture dishes. The cells were cultured without RA for an additional 2 days. A, expression patterns of Prp19 (upper panel) and Prp19 (lower panel) mRNAs. Total RNAs were prepared from P19 cells treated with RA for various times and analyzed by real-time PCR. B, expression levels of PRP19 and PRP19 proteins during neural differentiation. Cell lysates were prepared from RA-primed P19 cells and analyzed by Western blotting with polyclonal antibody against PRP19 (upper panel), PRP19 (middle panel), or actin (lower panel).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Isolation of Prp19 cDNA—To isolate the early response genes that participate in the RA-primed neural differentiation of mouse P19 cells, we employed a subtractive cDNA cloning strategy to enrich genes that are highly expressed in P19 cells after 24 h of RA treatment as described previously (16). Using the subtractive probes obtained, 9 x 10⁵ plaques from the rat brain gt11 cDNA library were screened, and nine clones whose expression levels were stimulated by >2-fold over the untreated control cells were identified. A homology search of the nine clones in the DDBJ, EBI, and GenBank^TM Data Banks showed that five were novel cDNA clones, and the remaining four clones were identified as acyl-CoA synthetase III, cytochrome c oxidase, Trip15/CSN2, and fibroblast growth factor receptor-2. In this study, we focused on one of the five novel clones (30-1-2) because the increment in its expression level was highest of the five novel clones during the RA-primed neural differentiation of P19 cells. To isolate the full-length 30-1-2 cDNA, we screened the rat brain gt10 cDNA library using the 1.1-kb EcoRI cDNA fragment obtained from the first screening as a probe and isolated the 1853-bp full-length cDNA clone. The cDNA encodes 504 amino acid residues, and its molecular mass is 55,238 Da. The cDNA sequence was deposited in DDBJ/GenBank^TM/EBI Data Bank under accession number AB020022 [GenBank] (Fig. 1A). A conserved domain search identified five WD40 repeat domains in the C-terminal region and a modified RING finger domain in the N-terminal region. A protein BLAST search in the GenBank^TM Data Bank revealed that the protein is identical or closely related to PRP19 in Mus musculus (100% identity), Homo sapiens (99.4%), and S. cerevisiae (36.0%) (Fig. 1B). Therefore, we designated the full-length 30-1-2 cDNA clone isolated from the rat brain cDNA library as rat Prp19. The deduced amino acid sequences of the rat and mouse PRP19 proteins are completely identical, suggesting that they could function in a similar manner. Thus, we used rat Prp19 cDNA throughout the experiment, even though P19 cells were originally established in a mouse embryo (36).

Detection of the PRP19 Splice Variant PRP19—Because Prp19 mRNA expression was up-regulated in the RA-primed neural differentiation of P19 cells, we analyzed the expression pattern of the Prp19 gene during the differentiation of mouse hippocampus-derived NS cells (Fig. 2). The NS cells formed neurospheres in medium containing 20 ng/ml EGF and actively incorporated bromodeoxyuridine (Fig. 2A, panel a) as described previously (18). When EGF-generated spheres were plated onto poly-L-lysine-coated tissue culture dishes after the removal of EGF and cultured in medium containing 20 ng/ml CNTF for 2 weeks, the NS cells differentiated into -tubulin III-positive neurons (Fig. 2A, panel b) and myelin basic protein-positive oligodendrocytes (panel c). The expression level of Prp19 mRNA during the differentiation of NS cells was analyzed by semiquantitative real-time PCR. In the presence of EGF, the expression level of Prp19 mRNA in NS cells was similar to that in untreated P19 cells, whereas the expression level in CNTF-treated NS cells was 1.5-fold higher than that in NS cells cultured with EGF (Fig. 2B). Furthermore, an additional PCR product termed Prp19 was detected in cDNA derived from CNTF-treated NS cells.

Sequence analysis revealed that this Prp19 cDNA possesses an additional 57 bp encoding 19 amino acid residues in-frame (DDBJ/Gen-Bank^TM/EBI accession number AB222602 [GenBank] ), located 245 bp downstream of the initiation codon of Prp19, and that its molecular mass is 57,301 Da (Fig. 2C). The chromosomal organization of exons encoding Prp19 was determined by alignment of the Prp19 cDNA in the mouse genome data base. The Prp19 gene was deduced to be composed of 16 exons with lengths ranging from 59 to 174 bp (Fig. 3A). Mouse genome sequence analysis revealed that the inserted sequence of Prp19 was derived from intron 3 by alternative splicing at a 3'-AG splice site and is located in-frame just before exon 4 (Fig. 3B). Both PRP19 and PRP19 possess three domains, including a modified RING finger domain, a nuclear localization signal, and WD40 repeat domains (Fig. 3C). To analyze the intracellular localization of proteins, pEGFP-PRP19 and pEGFP-PRP19 expression vectors were constructed and introduced into P19 cells (Fig. 3D). EGFP-fused PRP19 was detected in the nuclei, whereas EGFP-fused PRP19 was observed in the nuclei and cytoplasm. The same results were obtained by immunocytochemical analysis with anti-PRP19 and anti-PRP19 antibodies (data not shown).

View larger version (59K): [in this window] [in a new window]   FIGURE 5. Functional analysis of the Prp19 and Prp19 genes during neural differentiation. A, expression levels of PRP19 and PRP19 in transfectants. P19 cells were transfected with the empty pcDNA3-EF1-, pcDNA3-EF1--antisense Prp19, pcDNA3-EF1--sense Prp19, and pcDNA3-EF1--sense Prp19 expression vectors, and the stable transfectants obtained were designated V1, -A1, -S1, and -S1 cells, respectively. Cell lysates from these transfectants were analyzed by Western blotting with anti-PRP19 (upper panel), anti-PRP19 (middle panel), or anti-actin (lower panel) antibody. B, effects of sense and antisense Prp19 and sense Prp19 RNAs on neural differentiation. Aggregated V1 (panels a-d), -A1 (panels e-h), -S1 (panels i-l), and -S1 (panels m-p) cells were treated without (panels a, c, e, g, i, k, m, and o) or with (panels b, d, f, h, j, l, n, and p) RA for 4 days. At 4 and 7 days after replating, immunocytochemical analysis was performed using anti--tubulin III (upper panels) or anti-GFAP (lower panels) antibody. Scale bar = 100 µm. In the absence of RA, GFAP-positive astroglial cells were not observed in all transfectants. C and D, quantification of the effects of sense and antisense Prp19 and sense Prp19 RNAs on -tubulin III-positive neuronal differentiation without and with RA, respectively. -Tubulin III-positive neurons were counted in at least five fields/slide under a microscope. Each value is the mean ± S.E. of triplicate chamber slides. *, p < 0.014 compared with the V1 cells; **, p < 0.017; ***, p < 0.013. E, quantification of the effects of sense and antisense Prp19 and sense Prp19 RNAs on RA-primed GFAP-positive astroglial cell differentiation. GFAP-positive areas were measured in at least five fields/slide using Image Gauge software (Fujifilm, Tokyo). Each value is the mean ± S.E. of triplicate chamber slides. *, p < 0.002 compared with V1 cells; **, p <0.019.

Participation of PRP19 and PRP19 in Neural Differentiation—To demonstrate the participation of PRP19 and PRP19 in neural differentiation, we established P19 cell lines in which the exogenous sense and antisense Prp19 and sense Prp19 RNAs were constitutively expressed. The stable transfectants of the pcDNA3-EF1--antisense Prp19, pcDNA3-EF1--sense Prp19, and pcDNA3-EF1--sense Prp19 expression vectors were designated -A1, -S1 and -S1, respectively. The expression level of PRP19 in -A1 and -S1 cells was estimated to be 0.6- and 1.5-fold of that in empty vector-introduced V1 cells (Fig. 5A). The expression level of PRP19 in -S1 cells was 1.9-fold higher than that in V1 cells, whereas the expression level of PRP19 in -S1 and -A1 cells was similar to that in V1 cells.

View larger version (47K): [in this window] [in a new window]   FIGURE 6. Effects of the forced expression of sense and antisense Prp19 and sense Prp19 RNAs on the expression of neuronal and astroglial cell marker proteins. Aggregated V1, -A1, -S1, and -S1 cells were treated with or without RA for 4 days. A, the expression levels of -tubulin III and N-CAM 4 days after replating were analyzed by Western blotting with the respective antibodies. N-CAMs detected at 140, 160, and 180 kDa are specific markers of astrocytes, astrocytes/neurons, and neurons, respectively (71). B, the expression levels of GFAP 7 days after replating were analyzed by Western blotting with anti-GFAP antibody. In the absence of RA, the expression of GFAP was barely detected in any transfectants.

The effects of PRP19 and PRP19 on GFAP-positive astroglial cell differentiation were also examined. Although the differentiation of all transfectants to GFAP-positive astroglial cells was not observed without the addition of RA (Fig. 5B, panels c, g, k, and o), all transfectants differentiated into astroglial cells in the presence of RA (panels d, h, l, and p). The differentiation of -S1 cells to astroglial cells induced by RA was significantly increased (Fig. 5, B, panel p; and E), whereas the astroglial differentiation of -A1 cells was strikingly suppressed (Fig. 5, B, panel h; and E).

We further analyzed the effects of exogenous expression of PRP19 and PRP19 on the expression of neuronal and astroglial cell marker proteins by Western blotting with anti--tubulin III, anti-N-CAM, and anti-GFAP antibodies (Fig. 6). In aggregated -S1 cells, the expression levels of the neuronal markers -tubulin III and 180-kDa neuron-specific N-CAM were markedly induced without any addition of RA (Fig. 6A). The stimulatory effects of the forced expression of PRP19 on the neuronal markers in RA-primed -S1 cells were also observed. On the other hand, the expression of neuronal markers in -S1 and -A1 cells was reduced in the presence of RA. In all transfectants, the expression of GFAP, a marker of astroglial cells, was not observed without RA (Fig. 6B). In the presence of RA, the expression levels of GFAP in -S1 and -S1 cells were significantly enhanced. Conversely, the expression of GFAP in RA-primed -A1 cells was decreased.

These results were consistent with the data from immunocytochemical analysis. Thus, it seems that PRP19 plays a crucial role in the early stages of neural differentiation of P19 cells, whereas PRP19 acts as a stimulator of astroglial cell differentiation and conversely exhibits an inhibitory effect on neuronal differentiation.

Effect of Prp19-specific Antisense ODNs on Neural Differentiation—To further confirm the function of Prp19 in neural differentiation, we examined the effect of Prp19-specific antisense 15-mer ODNs on the neural differentiation of RA-primed P19 cells. When P19 cells were treated with 5 and 10 µM antisense Prp19 ODNs, the expression level of PRP19 protein was reduced to 40 and 30% of the level in untreated control cells, respectively (Fig. 7A). On the other hand, PRP19 expression levels in antisense ODN-treated cells were not affected. Immunocytochemical analysis revealed that, in the presence of the antisense ODNs, the neuronal differentiation of RA-primed P19 cells was dose-dependently stimulated, whereas astroglial cell differentiation was strikingly suppressed (Fig. 7, B-D). The inhibition observed in the presence of sense ODNs was perhaps due to the nonspecific inhibition of protein synthesis, as observed previously (33).

We also examined the effect of antisense ODNs on the expression levels of neuronal and astroglial cell markers in RA-primed P19 cells by Western blotting. The expression of the neuron-specific markers -tubulin III and 180-kDa neuron-specific N-CAM was increased in antisense ODN-treated cells compared with sense ODN-treated cells (Fig. 7E). In contrast, the expression level of GFAP in antisense ODN-treated cells was down-regulated (Fig. 7F). Thus, these results were consistent with the data from the forced expression of Prp19 RNA as described above.

Effect of PRP19 and PRP19 on Neurogenic Gene Expression—The data described above suggested that the forced expression of Prp19 RNA promoted both neurogenesis and gliogenesis, whereas the forced expression of Prp19 RNA promoted astroglial (but not neuronal) differentiation. To further explore the molecular mechanisms underlying the neural differentiation regulated by PRP19 and PRP19, we investigated the expression patterns of neural differentiation-related genes such as oct-3/4, nanog, Mash-1, and GFAP by Northern blotting. The transcription factor Oct-3/4 maintains the undifferentiated state of P19 cells as well as embryonic stem (ES) cells (37). The down-regulation of oct-3/4 mRNA is required for the onset of neural differentiation (16). On the basis of these facts, we analyzed the expression pattern of oct-3/4 mRNA during the neural differentiation of RA-primed V1, -A1, -S1, and -S1 cells (Fig. 8, A, upper panels; and F). The expression level of oct-3/4 mRNA in V1 cells was reduced to 50% of the original level after 28 h of RA treatment and became undetectable after 30 h. In contrast, the expression level of oct-3/4 mRNA in -S1 cells was lower than that in V1 cells and decreased to <50% of the original level after 20 h of RA treatment. Moreover, in aggregated -S1 cells (but not in aggregated V1 cells), the down-regulation of oct-3/4 mRNA expression occurred within 48 h in the absence of RA (Fig. 8, A, lower panels; and F). The decrease in the expression level of oct-3/4 mRNA was slowed in RA-primed -A1 and -S1 cells, and oct-3/4 mRNA expression was continued by at least 40 h.

Nanog is a recently discovered homeodomain transcription factor and is both sufficient and necessary for the maintenance of pluripotency of mouse ES cells and mouse epiblasts, independent of the leukemia inhibitory factor (LIF)/STAT3 (signal transducer and activator of transcription) signaling pathway (22, 23). We therefore analyzed the expression level of nanog mRNA during neural differentiation. Northern blot analysis revealed that nanog mRNA expression was induced in all aggregated transfectants regardless of treatment with RA, and its high level of expression was maintained until the onset of neural differentiation (Fig. 8B, upper panels). In RA-primed -S1 cells, the enhanced expression was lowered to a negligible level within 20 h. In contrast, in -S1 cells, substantial expression was detected even at 28 h after RA addition and became undetectable at 32 h. In V1 and -A1 cells, the enhanced expression disappeared after 24 h. In -S1 cells, the down-regulation of aggregation culture-induced nanog mRNA expression began after 20 h and reached a negligible level at 48 h without the addition of RA (Fig. 8, B, lower panels; and J). On the other hand, in the absence of RA, the down-regulation of nanog transcription was not observed in aggregated V1 cells. From these results, it seems likely that PRP19 participates in the down-regulation of oct-3/4 and nanog gene expression in the early stages of RA-primed neural differentiation of P19 cells, whereas PRP19 suppresses the down-regulation of the expression of these genes.

View larger version (47K): [in this window] [in a new window]   FIGURE 7. Effect of antisense Prp19 ODNs on neural differentiation. A, expression levels of PRP19 and PRP19 in antisense Prp19 ODN-treated cells. P19 cells were treated with various concentrations of the ODNs for 48 h, and the expression levels of PRP19, PRP19, and actin were analyzed by Western blotting with the respective antibodies. B, effect of antisense Prp19 ODNs on -tubulin III-positive neuronal and GFAP-positive astroglial cell differentiation. Aggregated P19 cells were treated with various concentrations of sense and antisense Prp19 ODNs in the presence of RA for 4 days. Immunocytochemical analyses with anti--tubulin III and anti-GFAP antibodies were performed 4 and 7 days after replating, respectively. Scale bar = 30 µm. C, quantification of the effect of antisense Prp19 ODNs on -tubulin III-positive neurogenesis. -Tubulin III-positive neurons were counted in at least five fields/slide under a microscope. Each value is the mean ± S.E. of triplicate chamber slides. *, p < 0.02 compared with the control. D, quantification of the effect of antisense Prp19 ODNs on GFAP-positive astrogliogenesis. GFAP-positive areas were measured in at least five fields/slide using Image Gauge software. Each value is the mean ± S.E. of triplicate chamber slides. *, p < 0.01 compared with 10 µM sense ODNs. E, effect of antisense Prp19 ODNs on the expression of neuronal marker proteins. The expression levels of -tubulin III and N-CAM 4 days after replating were analyzed by Western blotting with the respective antibodies. 180-kDa N-CAM is a neuron-specific marker. F, effect of antisense Prp19 ODNs on the expression of astroglial marker proteins. The expression levels of GFAP 7 days after replating were analyzed by Western blotting with anti-GFAP antibody.

View larger version (74K): [in this window] [in a new window]   FIGURE 8. Effects of PRP19 and PRP19 on various gene expressions participating in neural differentiation. V1, -A1, -S1, and -S1 cells were cultured with or without RA for various times. The expression levels of oct-3/4 (A), nanog (B), Mash-1 (C), and GFAP (D) mRNAs were analyzed by Northern blotting. Ribosomal phosphoprotein (PO) mRNA (E) was analyzed simultaneously as an internal control. The relative expression levels of oct-3/4 (F), Mash-1 (G), and GFAP (H) mRNAs were determined during the RA-primed neural differentiation of V1, -A1, -S1, and -S1 cells, and those of oct-3/4 (I), nanog (J), and Mash-1 (K) mRNAs were determined during neural differentiation induced by aggregation culture without RA. The relative ratios of the mRNA expression levels were estimated compared with the respective maximal expression levels in each cell line. The expression level of ribosomal phosphoprotein mRNA was used as an internal control. The values indicate the average of two independent experiments.

View larger version (34K): [in this window] [in a new window]   FIGURE 9. PRP19 (but not PRP19) is a component of the spliceosome in P19 cells. A, coexistence of PRP19 with the major spliceosome components. Cell lysates were prepared from P19 cells treated with RA for 0 and 24 h and then immunoprecipitated (IP) with anti-PRP19 or anti-PRP19 antibody. The resulting immunoprecipitates were subjected to 10% SDS-PAGE and analyzed by Western blotting (WB) with anti-PSF and anti-CDC5L antibodies. B, gel filtration analysis of the spliceosome. Cell lysates prepared from P19 cells treated with RA for 0 and 24 h were fractionated by Sepharose 4B column chromatography. Each fraction was analyzed by Western blotting with anti-PRP19, anti-CDC5L, and anti-PRP19 antibodies to determine the elution profiles of the respective proteins. Blue dextran 2000 (2000 kDa), thyroglobulin (670 kDa), apoferritin (480 kDa), -globulin (160 kDa), and bovine serum albumin (66 kDa) were used as size markers. C, yeast two-hybrid analysis of the interaction between PRP19 and CDC5L. Prp19 deletion constructs were used to identify the binding region of PRP19 with CDC5L by yeast two-hybrid analysis. CDC5L bound to the N-terminal region (N) of PRP19 (amino acids 1-166), but not the middle region (M; amino acids 166-368), the C-terminal region (C; amino acids 368-504), and the region from M to C (M+C; amino acids 166-504). NLS indicates nuclear localization signal. ++, strong interaction as scored by growth on Leu-/Trp-/His- medium and quantitative -galactosidase assays; +, weak interaction; -, no interaction.

Identification of PRP19-specific Binding Proteins—To further analyze the role of PRP19 in the process of neural differentiation, cDNA clones encoding mouse PRP19-binding proteins were searched using a yeast two-hybrid screening system. Yeast strain Y153 (Leu^-/Trp^-/His^-) was cotransfected with a bait vector expressing a fusion protein composed of the yeast Gal4 DNA-binding domain and sequence 83-101 of mouse PRP19 and with an RA-treated P19 cell cDNA library directing the synthesis of fusion proteins composed of cDNA-encoded proteins and the Gal4 transcriptional activation domain (Fig. 10A). Strain Y153 contains the HIS3 and lacZ genes linked to the GAL4 promoter. Upon transfection of 1.3 x 10⁶ Y153 cells, 38 His⁺ clones were developed, and 10 of the 38 clones were -galactosidase-positive. Sequence analysis of the clones showed that three of the 10 clones were novel cDNA clones, and six were identified as known cDNA clones, such as CypA; prothymosin-; endometrial bleeding-associated factor; proteasome activator subunit-4; and ribosomal proteins S7, S15a, and S26. Among them, CypA is specifically expressed in neurons and participates in the neuronal (but not astroglial) differentiation of P19 cells (44, 45). Therefore, in this study, we focused on the functional analysis of CypA in neural differentiation regulated by PRP19 and PRP19. Yeast two-hybrid analysis showed that the isolated CypA cDNA encoding amino acids 61-163 specifically bound to the N-terminal region (amino acids 83-101) of PRP19, although association with full-length PRP19 was not detected (Fig. 10, A and B).

P19 cells were transfected with the pcDNA3-EF1--HA-CypA plasmid vector to express HA-tagged full-length CypA and cultured for 24 h. The cells were then treated with RA for an additional 24 h and subjected to Western blot analysis. Significant expression of HA-tagged CypA and PRP19 was observed (Fig. 10C). Immunoprecipitation of the extract from the cells with anti-HA antibody revealed that CypA coprecipitated with PRP19. In the absence of RA, coprecipitation was not detected, as in the vacant vector-transfected cells. These results imply that the conformational change in PRP19 after RA treatment contributes to the association with CypA.

Involvement of CypA in Neurogenesis—To confirm the participation of CypA in neuronal differentiation, we constructed pcDNA3-EF1--sense CypA and pcDNA3-EF1--antisense CypA expression vectors and introduced them into P19 cells. The resulting stable transfectants were designated CypA-S1 and CypA-A1 cells, respectively. The expression levels of CypA-S1 and CypA-A1 cells were estimated to be 1.32- and 0.68-fold of that of empty vector-transfected V1 cells, respectively (Fig. 11A). In the absence of RA, CypA-S1 cells differentiated into -tubulin III-positive neurons after the simple aggregation culture, as observed in -S1 cells (Fig. 11, B, panel d; and C). In the presence of RA, the neuronal differentiation of CypA-S1 cells was enhanced, whereas the neurogenesis of CypA-A1 cells was vigorously suppressed compared with V1 cells (Fig. 11, B, panels e and h; and D). Although the effect of sense CypA on astroglial cell differentiation was not evident in the presence of RA, the appearance of GFAP-positive astroglia in CypA-A1 cells was strongly suppressed (Fig. 11 B, panels f and i; and E). Thus, these results are consistent with a previous report except for the suppression of gliogenesis (45), suggesting that CypA participates in the early stages of neural differentiation.

View larger version (30K): [in this window] [in a new window]   FIGURE 10. Interaction between CypA and PRP19. A, schematic illustration of cDNA inserted into vectors used for yeast two-hybrid analysis. Various fragments of Prp19 and Prp19 cDNAs were ligated in-frame with the Gal4 DNA-binding domain (GAL4DBD) in pAS2-1 as bait. CypA cDNA corresponding to amino acid residues 61-165 (which was identified as the binding region with PRP19 in the first screening) was ligated with the Gal4 activation domain (GAL4AD) into pACT2. NLS, nuclear localization signal. B, yeast two-hybrid analysis of the interaction between CypA and PRP19. The CypA and PRP19 expression constructs were simultaneously introduced into yeast strain Y153. The cells were streaked on selection medium (Leu-/Trp-/His- (-LWH)/3-aminotriazole+). posi and nega indicate a positive control (combination of pAS2-1-PRP19 and pACT2-KIAA0907) and a negative control (combination of pAS2-1-PRP19-(83-101) and pACT2), respectively. C, interaction of CypA with endogenous PRP19 in P19 cells. P19 cells were transfected with the pcDNA3-EF1--HA or pcDNA3-EF1--HA-CypA vector and then cultured with or without RA for 24 h. The cell lysates were prepared, immunoprecipitated (IP) with anti-HA antibody, and subjected to Western blotting (WB) with anti-PRP19 (upper panel) or anti-HA (lower panel) antibody.

PRP19 Suppresses CypA-dependent Activation of RAREoct Promoter Activity—CypA possesses a peptidylprolyl isomerase activity and is a target of the immunosuppressive drug cyclosporin A (44). Therefore, we investigated the effect of CypA on the RAREoct promoter activity using the CypA expression vector and the pLuc-RAREoct reporter plasmid (Fig. 13A). In RA-primed P19 cells, CypA enhanced RAREoct-mediated gene expression in a dose-dependent manner (Fig. 13B, bars 1-3). The activation of gene expression by CypA was completely inhibited by the addition of cyclosporin A (bars 10 and 11), confirming that CypA can activate RAREoct-mediated gene expression. We also examined the effect of PRP19 on the CypA-dependent RAREoct promoter activity. RAREoct-mediated gene expression by CypA was decreased by cotransfection with the PRP19 expression vector (bars 4-7), although no effect on the basal level of RAREoct promoter activity by PRP19 was observed. To further define the role of RAR and CypA in the activation of the oct-3/4 promoter, we analyzed soluble chromatin from P19 cells treated with or without RA for 6 h by the chromatin immunoprecipitation assay (Fig. 13C). We found that RAR and CypA were present on the oct-3/4 promoter in the absence of RA. After 6 h of RA treatment, robust acetylation of histone H3 was observed, suggesting that the recruitment of the transcriptional coactivator is caused by the CypA-dependent conformational change in RAR, possibly the RAR/RXR heterodimer, in the presence of RA. Thus, these results imply that PRP19 inhibits CypA-dependent oct-3/4 gene expression via direct binding with CypA.

View larger version (72K): [in this window] [in a new window]   FIGURE 11. Effects of the enforced expression of sense and antisense CypA RNAs on neural differentiation. A, expression levels of CypA protein in vector-transfected P19 cells. P19 cells were transfected with the vacant pcDNA3-EF1-, pcDNA3-EF1--sense CypA, and pcDNA3-EF1--antisense CypA expression vectors, and the resulting transfectants were designated V1, CypA-S1, and CypA-A1 cells, respectively. Cell lysates from these transfectants were analyzed by Western blotting with anti-CypA antibody. B, effects of the expression of sense and antisense CypA RNAs on neural differentiation. Aggregated V1 (panels a-c), CypA-S1 (panels d-f), and CypA-A1 (panels g-i) cells were cultured without (panels a, d, and g) or with (panels b, c, e, f, h, and i) RA for 4 days. Immunocytochemical analysis was performed 4 and 7 days after replating using anti--tubulin III (upper and middle panel) or anti-GFAP (lower panel) antibody. Fluorescein isothiocyanate-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) was used as a secondary antibody. Nuclei were stained with Hoechst 33258. Scale bar = 100 µm. C and D, quantification of the effects of sense and antisense CypA RNAs on -tubulin III-positive neuronal differentiation. -Tubulin III-positive neurons in the absence (C) or presence (D) of RA were counted in at least five fields/slide under an Axioplan 2 fluorescence microscope. Each value is the mean ± S.E. of triplicate chamber slides. *, p < 0.001 compared with V1 cells. E, quantification of the effects of sense and antisense CypA RNAs on RA-primed GFAP-positive astroglial cell differentiation. GFAP-positive areas were counted in at least five fields/slide under an Axioplan 2 fluorescence microscope. Each value is the mean ± S.E. of triplicate chamber slides. *, p < 0.03 compared with V1 cells.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Critical levels of oct-3/4 and nanog gene expression are required for the maintenance of pluripotency of ES cells (48). A <2-fold increase in expression causes differentiation into the endoderm and mesoderm, whereas reduction to <50% of the normal expression level triggers differentiation into the trophectoderm (49). During stroma cell-derived inducing activity-mediated neurogenesis of ES cells, the up-regulation of oct-3/4 mRNA expression is sustained (47). Suppression of oct-3/4 mRNA expression abolishes neuronal differentiation even after stimulation by stroma cell-derived inducing activity. Likewise, nanog-deficient ES cells lose pluripotency and differentiate into the extraembryonic endoderm lineage (22); in P19 cells, Oct-3/4 protein is also present, but is absent in all differentiated somatic cell types in vivo and in vitro (21). In this study, we found that the constitutive expression of Prp19 RNA in P19 cells caused the up-regulation of oct-3/4 and nanog mRNA expression within 12 h of RA treatment and that the expression of these genes was thereafter rapidly down-regulated (Fig. 8, A and B). Furthermore, the induction of the neurogenic bHLH transcription factor Mash-1 in -S1 cells was more rapid and intense than that in V1 cells. Yeast two-hybrid and co-immunoprecipitation analyses showed that PRP19 could directly bind to the spliceosome components PSF and CDC5L (Fig. 9). These results support the idea that, downstream of the signal evoked by a simple aggregation culture, PRP19 participates in the down-regulation of genes that maintain the multipotency of P19 cells.

View larger version (54K): [in this window] [in a new window]   FIGURE 12. Change in gene expression during neural differentiation in RA-primed P19 cells. Total RNAs were extracted from P19 cells treated with RA for various times, and the expression levels of CypA mRNA (A) and oct-3/4, otx2, COUP-TFI, RAR, and RAR mRNAs (B) were analyzed by Northern blotting. Images of ethidium bromide-stained 28 S ribosomal RNA are indicated for comparison with the total amount of RNA employed.

As in neurogenesis in vivo, neurons appear earlier than astroglial cells during RA-primed P19 cell neural differentiation (36). Neurogenic bHLH transcription factors promote neuronal determination (55, 56). Comparable bHLH factors that promote the formation of astrocytes have not been isolated. Nonetheless, recent studies have shown that changes in the expression levels of neurogenic bHLH genes might be an important event in the switch from neurogenesis to gliogenesis. In Mash-1/Math-3 or Mash-2/Ngn-2 double knockout mice, neurogenesis is blocked at the precursor stage, and astrocytes are excessively produced (20, 57). Ngn-1 promotes neurogenesis via the Ngn-1·cAMP-responsive element-binding protein-binding protein·p300 complex and inhibits astrocyte differentiation by sequestering the cAMP-responsive element-binding protein-binding protein·Smad transcription complex away from astrocyte differentiation genes and by inhibiting the activation of STAT transcription factors (58). In Drosophila, neuronal and longitudinal glial fates are determined by the gcm gene. In gcm-deficient flies, glial cells are converted to neurons, whereas ectopic expression of gcm in neurons causes neuron-glial transformation (1, 59). Two mammalian homologs (Gcm1 and Gcm2) have been identified (2-6). However, Gcm1 exhibits only the potential to induce gliogenesis, but may function in the generation of a minor subpopulation of glial cells (10). Thus, in mammals, the genes that might control a GCM-like switching mechanism have not yet identified.

In this situation, we found that PRP19 could function as a neuronglial switch. How does PRP19 contribute to a glial cell fate decision? Yeast two-hybrid analysis showed that CypA is one of the PRP19-specific binding partners. The overexpression of CypA RNA in P19 cells was no longer required for RA treatment to initiate neurogenesis, but not gliogenesis. RAREoct-mediated reporter assay indicated that CypA could function as a transcriptional activator through the conformational change in the liganded RAR/RXR heterodimer and that its activity was repressed by PRP19 via binding with CypA on the oct-3/4 promoter (Figs. 13 and 14). Collectively, it is possible that, within 12 h of the immediate-early stages of neuronal differentiation defined by the transient expression of the neuroectodermal marker otx2, the sustained enhancement of oct-3/4 expression by CypA was essentially required for neurogenesis and that the expression of oct-3/4 was subsequently reduced by the following transient induction of PRP19 expression (Figs. 12 and 14). It has also been reported that, in P19 cells, knockdown of CypA by RNA interference causes loss of RA-primed neuronal differentiation, but not dimethyl sulfoxide-induced mesodermal differentiation, and a marked reduction of RA-induced RARE-mediated reporter activity, showing that CypA promotes RARE-mediated neurogenic gene expression (45). Thus, the enhancement of astrocytogenesis by the forced expression of Prp19 RNA might be due at least in part to the inhibition of neurogenesis.

LIF-induced Jak (Janus kinase)/STAT signaling is a critical part of the astrogliogenic machinery (15, 60). Mouse knockout studies have demonstrated a genetic deficiency in major components of this pathway, including LIF, its receptors LIF receptor- and gp130, and the signaling molecules STAT1 and STAT3 (61-63). In addition, factors that promote astrogliogenesis, including BMPs, basic fibroblast growth factor, and Notch signaling, all require pre-activation of the Jak/STAT pathway (64-67). Furthermore, the forced expression of the EGF receptor in early progenitors induces precocious astrocyte differentiation in response to LIF by increasing STAT3 protein levels (68), showing that, during the neurogenic period, the Jak/STAT pathway is only weakly activated. Recently, He et al. (41) reported a positive autoregulatory loop of the Jak/STAT pathway, by which STAT1/3 directly activates the expression of many components of this pathway. This feed-forward loop provides a positive mechanism to direct the progenitor stem cells toward astrogliogenesis, showing that sufficient activation of the Jak/STAT pathway function is a switch for transition from neurogenesis to gliogenesis. During the neural differentiation of aggregated P19 cells in the presence of RA, EGF receptor and BMP4 mRNA expression is markedly up-regulated (25, 69). We observed that both aggregation and RA treatments were required for the astroglial cell differentiation of -S1 cells, in which sense Prp19 RNA was overexpressed (Fig. 5). In a preliminary experiment, we also investigated the phosphorylation level of STAT3 during the RA-primed neural differentiation of -S1 cells using anti-Tyr(P)⁷⁰⁵ STAT3 antibody. The phosphorylation of STAT3 in -S1 cells could be detected after 3 days of RA treatment, when the phosphorylation in the other transfectants, including V1, -A1, and -S1 cells, could not (data not shown). Thus, these data strongly imply that only PRP19 overexpression is insufficient for the functional activation of the Jak/STAT pathway. Probably, PRP19 participates in the sufficient activation of the Jak/STAT pathway in cooperation (at least in part) with the signals evoked by LIF and BMPs to direct P19 cells toward astrogliogenesis.

View larger version (27K): [in this window] [in a new window]   FIGURE 13. Inhibitory effect of PRP19 on CypA-dependentoct-3/4promoter activity.A,schematicillustration of the luciferase (Luc) reporter plasmid linked to the RAREoct promoter. The promoter region of oct-3/4 (-412 to +39) was introduced into the pTRE2-Lucvector, yielding pLuc-RAREoct. The recognition sequence of RAR in theoct-3/4promoter(-48 to -28)is also indicated (46). B, suppression of CypA-dependent RAREoct promoter activity by PRP19.P19 cells were transfected with 1 µg of pLuc-RAREoct and 0.5 µg of pcDNA3.1/Myc-His/LacZ together with a total of 1 µg of pcDNA3-EF1--sense CypA, pcDNA3-EF1--sense Prp19, and empty pcDNA3-EF1- expression vectors in various combinations by lipofection. After 24 h of transfection, the cells were treated with RA for 24 h, and luciferase activities in the cell lysates were determined according to the recommendations of Promega Corp. and normalized based on -galactosidase activity as an internal control. The effect of cyclosporin A (CsA) was also determined. Each value is the mean ± S.E. of triplicate culture dishes. *,p<0.002 compared with 0.25 µg of CypA expression vector; **,p<0.002 compared with 0.5 µg of CypA expression vector. C, occupancy of the oct-3/4 promoter by RAR and CypA. Soluble chromatins were prepared from P19 cells treated with or without RA for 6 h and immunoprecipitated with anti-RAR or anti-CypA antibody. The DNAs extracted from the immunoprecipitates were amplified using primer pairs covering the oct-3/4 promoter (-412 to +39). The acetylation of histone H3 (AcH3) was simultaneously analyzed.

View larger version (21K): [in this window] [in a new window]   FIGURE 14. Model of the roles of PRP19 and PRP19 in neural differentiation. The functions of PRP19 and PRP19 are distinct from each other. In the immediate-early stages of neural differentiation of RA-primed P19 cells, Prp19 gene expression is up-regulated to induce oct-3/4 gene expression, by which P19 cells are committed to neuroectodermal lineage cells. In the late-early stages, PRP19 differentiates neuroectodermal cells to NS cells through the COUP-TFI-dependent down-regulation of oct-3/4 mRNA expression. PRP19 also participates in this step. Thereafter, PRP19 promotes neuronal differentiation in cooperation (at least in part) with Wnt-1, whose expression could be up-regulated by cell aggregation alone. In contrast, PRP19 inhibits neurogenesis and inversely stimulates astrogliogenesis during the induction of neuronal and glial/astroglial progenitor cells from the NS cells, respectively. Presumably, PRP19 promotes astrogliogenesis via stimulation of the Jak/STAT pathway in cooperation with the signals evoked by autocrine/paracrine factors, including BMPs. In the immediate-early stage, CypA enhances the RAREoct promoter activity via the activation of the liganded RAR/RXR heterodimer and leads to the differentiation of ES cells to neuroectodermal cells. In the late-early stage, PRP19 inhibits oct-3/4 gene expression through direct binding with CypA. Thereafter, the RAR·RXR complex existing on the RAREoct promoter is replaced with the COUP-TFI homodimer, which conducts the complete suppression of oct-3/4 gene expression.

	FOOTNOTES

 
The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/Gen-Bank^TM/EBI Data Bank with accession number(s) AB020022 [GenBank] and AB222602 [GenBank] .

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 81-4-7124-1501; Fax: 81-4-7125-1841; E-mail: ftashir{at}rs.noda.tus.ac.jp.

² The abbreviations used are: BMP, bone morphogenetic protein; CNTF, ciliary neurotrophic factor; RA, retinoic acid; bHLH, basic helix-loop-helix; GFAP; glial fibrillary acidic protein; ODNs, oligodeoxynucleotides; NS, neural stem; EGF, epidermal growth factor; RAR, retinoic acid receptor; N-CAM, neural cell adhesion molecule; PSF, polypyrimidine tract-binding protein-associated splicing factor; EGFP, enhanced green fluorescent protein; HA, hemagglutinin; RARE, retinoic acid-responsive element; ES, embryonic stem; LIF, leukemia inhibitory factor; COUP-TFI, chicken ovalbumin upstream transcription factor I; RXR, retinoid X receptor.

	ACKNOWLEDGMENTS

 
We thank Profs. K. Oda and A. I. Lamond for encouragement throughout this study and for providing anti-CDC5L antibody, respectively.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Hosoya, T., Takizawa, K., Nitta, K., and Hotta, Y. (1995) Cell 82, 1025-1036[CrossRef][Medline] [Order article via Infotrieve]
2. Akiyama, Y., Hosoya, T., Poole, A. M., and Hotta, Y. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 14912-14916[Abstract/Free Full Text]
3. Altshuller, Y., Copeland, N. G., Gilbert, D. J., Jenkins, N. A., and Frohman, M. A. (1996) FEBS Lett. 393, 201-204[CrossRef][Medline] [Order article via Infotrieve]
4. Kammerer, M., Pirola, B., Giglio, S., and Giangrande, A. (1999) Cytogenet. Cell Genet. 84, 43-47[CrossRef][Medline] [Order article via Infotrieve]
5. Kanemura, Y., Hiraga, S., Arita, N., Ohnishi, T., Izumoto, S., Mori, K., Matsumura, H., Yamasaki, M., Fushiki, S., and Yoshimine, T. (1999) FEBS Lett. 442, 151-156[CrossRef][Medline] [Order article via Infotrieve]
6. Kim, J., Jones, B. W., Zock, C., Chen, Z., Wang, H., Goodman, C. S., and Anderson, D. J. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 12364-12369[Abstract/Free Full Text]
7. Basyuk, E., Cross, J. C., Corbin, J., Nakayama, H., and Hunter, P. (1999) Dev. Dyn. 214, 303-311[CrossRef][Medline] [Order article via Infotrieve]
8. Anson-Cartwright, L., Dawson, K., Holmyard, D., Fisher, S. J., Lazzarini, R. A., and Cross, J. C. (2000) Nat. Genet. 25, 311-314[CrossRef][Medline] [Order article via Infotrieve]
9. Schreiber, J., Riethmacher-Sonnenberg, E., Riethmacher, D., Tuerk, E. E., Enderich, J., Bosl, M. R., and Wegner, M. (2000) Mol. Cell. Biol. 20, 2466-2474[Abstract/Free Full Text]
10. Iwasaki, Y., Hosoya, T., Takebayashi, H., Ogawa, Y., Hotta, Y., and Ikenaka, K. (2003) Development (Camb.) 130, 6027-6035[Abstract/Free Full Text]
11. Gunther, T., Chen, Z. F., Kim, J., Priemel, M., Rueger, J. M., Amling, M., Moseley, J. M., Martin, T. J., Anderson, D. J., and Karsenty, G. (2000) Nature 406, 199-203[CrossRef][Medline] [Order article via Infotrieve]
12. Kim, J., Lo, L., Dormand, E., and Anderson, D. J. (2003) Neuron 38, 17-31[CrossRef][Medline] [Order article via Infotrieve]
13. Stolt, C. C., Lommes, P., Sock, E., Chaboissier, M. C., Schedl, A., and Wegner, M. (2003) Genes Dev. 17, 1677-1689[Abstract/Free Full Text]
14. Hardy, R. J. (1998) J. Neurosci. Res. 54, 46-57[CrossRef][Medline] [Order article via Infotrieve]
15. Johe, K. K., Hazel, T. G., Muller, T., Dugich-Djordjevic, M. M., and McKay, R. D. (1996) Genes Dev. 10, 3129-3140[Abstract/Free Full Text]
16. Akiyama, H., Sugiyama, A., Uzawa, K., Fujisawa, N., Tashiro, Y., and Tashiro, F. (2003) Brain Res. Dev. Brain Res. 140, 45-56[Medline] [Order article via Infotrieve]
17. Cheng, S. C., Tarn, W. Y., Tsao, T. Y., and Abelson, J. (1993) Mol. Cell. Biol. 13, 1876-1882[Abstract/Free Full Text]
18. Mukobata, S., Urano, Y., Hibino, T., Sugawara, T., Sugiyama, A., and Tashiro, F. (2000) Res. Commun. Biochem. Cell Mol. Biol. 4, 221-234
19. Fort, P., Marty, L., Piechaczyk, M., el Sabrouty, S., Dani, C., Jeanteur, P., and Blanchard, J. M. (1985) Nucleic Acids Res. 13, 1431-1442[Abstract/Free Full Text]
20. Nieto, M., Schuurmans, C., Britz, O., and Guillemot, F. (2001) Neuron 29, 401-413[CrossRef][Medline] [Order article via Infotrieve]
21. Okamoto, K., Okazawa, H., Okuda, A., Sakai, M., Muramatsu, M., and Hamada, H. (1990) Cell 60, 461-472[CrossRef][Medline] [Order article via Infotrieve]
22. Mitsui, K., Tokuzawa, Y., Itoh, H., Segawa, K., Murakami, M., Takahashi, K., Maruyama, M., Maeda, M., and Yamanaka, S. (2003) Cell 113, 631-642[CrossRef][Medline] [Order article via Infotrieve]
23. Chambers, I., Colby, D., Robertson, M., Nichols, J., Lee, S., Tweedie, S., and Smith, A. (2003) Cell 113, 643-655[CrossRef][Medline] [Order article via Infotrieve]
24. Franco del Amo, F., Gendron-Maguire, M., Swiatek, P. J., and Gridley, T. (1993) Biochim. Biophys. Acta 1171, 323-327[Medline] [Order article via Infotrieve]
25. Tang, K., Yang, J., Gao, X., Wang, C., Liu, L., Kitani, H., Atsumi, T., and Jing, N. (2002) Biochem. Biophys. Res. Commun. 293, 167-173[CrossRef][Medline] [Order article via Infotrieve]
26. Takahashi, J., Palmer, T. D., and Gage, F. H. (1999) J. Neurobiol. 38, 65-81[CrossRef][Medline] [Order article via Infotrieve]
27. Simeone, A., Acampora, D., Gulisano, M., Stornaiuolo, A., and Boncinelli, E. (1992) Nature 358, 687-690[CrossRef][Medline] [Order article via Infotrieve]
28. Strausberg, R. L., et al. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 16899-16903[Abstract/Free Full Text]
29. Zelent, A., Mendelsohn, C., Kastner, P., Krust, A., Garnier, J. M., Ruffenach, F., Leroy, P., and Chambon, P. (1991) EMBO J. 10, 71-81[Medline] [Order article via Infotrieve]
30. Hasel, K. W., and Sutcliffe, J. G. (1990) Nucleic Acids Res. 18, 4019[Free Full Text]
31. Boudjelal, M., Taneja, R., Matsubara, S., Bouillet, P., Dolle, P., and Chambon, P. (1997) Genes Dev. 11, 2052-2065[Abstract/Free Full Text]
32. Ajuh, P., Kuster, B., Panov, K., Zomerdijk, J. C., Mann, M., and Lamond, A. I. (2000) EMBO J. 19, 6569-6581[CrossRef][Medline] [Order article via Infotrieve]
33. Sugiyama, A., Miyagi, Y., Komiya, Y., Kurabe, N., Kitanaka, C., Kato, N., Nagashima, Y., Kuchino, Y., and Tashiro, F. (2003) Carcinogenesis 24, 1549-1559[Abstract/Free Full Text]
34. Akiyama, H., Fujisawa, N., Tashiro, Y., Takanabe, N., Sugiyama, A., and Tashiro, F. (2003) J. Biol. Chem. 278, 10752-10762[Abstract/Free Full Text]
35. Pikarsky, E., Sharir, H., Ben-Shushan, E., and Bergman, Y. (1994) Mol. Cell. Biol. 14, 1026-1038[Abstract/Free Full Text]
36. Jones-Villeneuve, E. M., McBurney, M. W., Rogers, K. A., and Kalnins, V. I. (1982) J. Cell Biol. 94, 253-262[Abstract/Free Full Text]
37. Shimazaki, T., Okazawa, H., Fujii, H., Ikeda, M., Tamai, K., McKay, R. D., Muramatsu, M., and Hamada, H. (1993) EMBO J. 12, 4489-4498[Medline] [Order article via Infotrieve]
38. Johnson, J. E., Zimmerman, K., Saito, T., and Anderson, D. J. (1992) Development (Camb.) 114, 75-87[Abstract]
39. McCormick, M. B., Tamimi, R. M., Snider, L., Asakura, A., Bergstrom, D., and Tapscott, S. J. (1996) Mol. Cell. Biol. 16, 5792-5800[Abstract]
40. Massari, M. E., and Murre, C. (2000) Mol. Cell. Biol. 20, 429-440[Free Full Text]
41. He, F., Ge, W., Martinowich, K., Becker-Catania, S., Coskun, V., Zhu, W., Wu, H., Castro, D., Guillemot, F., Fan, G., Vellis, J., and Sun, Y. (2005) Nat. Neurosci. 8, 616-625[CrossRef][Medline] [Order article via Infotrieve]
42. Makarov, E. M., Makarova, O. V., Urlaub, H., Gentzel, M., Will, C. L., Wilm, M., and Luhrmann, R. (2002) Science 298, 2205-2208[Abstract/Free Full Text]
43. Deleted in proof
44. Goldner, F. M., and Patrick, J. W. (1996) J. Comp. Neurol. 372, 283-293[CrossRef][Medline] [Order article via Infotrieve]
45. Song, J., Lu, Y.-C., Yokoyama, K., Rossi, J., and Chiu, R. (2004) J. Biol. Chem. 279, 24414-24419[Abstract/Free Full Text]
46. Ben-Shushan, E., Sharir, H., Pikarsky, E., and Bergman, Y. (1995) Mol. Cell. Biol. 15, 1034-1048[Abstract]
47. Shimozaki, K., Nakashima, K., Niwa, H., and Taga, T. (2003) Development (Camb.) 130, 2505-2512[Abstract/Free Full Text]
48. Deleted in Proof
49. Niwa, H., Miyazaki, J., and Smith, A. G. (2000) Nat. Genet. 24, 372-376[CrossRef][Medline] [Order article via Infotrieve]
50. Schmidt, J. W., Brugge, J. S., and Nelson, W. J. (1992) J. Cell Biol. 116, 1019-1133[Abstract/Free Full Text]
51. Teramoto, S., Kihara-Negishi, F., Sakurai, T., Yamada, T., Hashimoto-Tamaoki, T., Tamura, S., Kohno, S., and Oikawa, T. (2005) Oncol. Rep. 14, 1231-1238[Medline] [Order article via Infotrieve]
52. Hirabayashi, Y., Itoh, Y., Tabata, H., Nakajima, K., Akiyama, T., Masuyama, N., and Gotoh, Y. (2004) Development (Camb.) 131, 2791-2801[Abstract/Free Full Text]
53. Duncan, M. K., Bordas, L., Dicicco-Bloom, E., and Chada, K. K. (1997) Dev. Dyn. 208, 107-114[CrossRef][Medline] [Order article via Infotrieve]
54. Benezra, R., Davis, R. L., Lockshon, D., Turner, D. L., and Weintraub, H. (1990) Cell 61, 49-59[CrossRef][Medline] [Order article via Infotrieve]
55. Lee, J. E. (1997) Curr. Opin. Neurobiol. 7, 13-20[CrossRef][Medline] [Order article via Infotrieve]
56. Cai, L., Morrow, E. M., and Cepko, C. L. (2000) Development (Camb.) 127, 3021-3030[Abstract]
57. Tomita, K., Moriyoshi, K., Nakanishi, S., Guillemot, F., and Kageyama, R. (2000) EMBO J. 19, 5460-5472[CrossRef][Medline] [Order article via Infotrieve]
58. Sun, Y., Nadal-Vicens, M., Misono, S., Lin, M. Z., Zubiaga, A., Hua, X., Fan, G., and Greenberg, M. E. (2001) Cell 104, 365-376[CrossRef][Medline] [Order article via Infotrieve]
59. Vincent, S., Vonesch, J. L., and Giangrande, A. (1996) Development (Camb.) 122, 131-139[Abstract]
60. Bonni, A., Sun, Y., Nadal-Vicens, M., Bhatt, A., Frank, D. A., Rozovsky, I., Stahl, N., Yancopoulos, G. D., and Greenberg, M. E. (1997) Science 278, 477-483[Abstract/Free Full Text]
61. Bugga, L., Gadient, R. A., Kwan, K., Stewart, C. L., and Patterson, P. H. (1998) J. Neurobiol. 36, 509-524[CrossRef][Medline] [Order article via Infotrieve]
62. Koblar, S. A., Turnley, A. M., Classon, B. J., Reid, K. L., Ware, C. B., Cheema, S. S., Murphy, M., and Bartlett, P. F. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 3178-3181[Abstract/Free Full Text]
63. Nakashima, K., Wiese, S., Yanagisawa, M., Arakawa, H., Kimura, N., Hisatsune, T., Yoshida, K., Kishimoto, T., Sendtner, M., and Taga, T. (1999) J. Neurosci. 19, 5429-5434[Abstract/Free Full Text]
64. Ge, W., Martinowich, K., Wu, X., He, F., Miyamoto, A., Fan, G., Weinmaster, G., and Sun, Y. E. (2002) J. Neurosci. Res. 69, 848-860[CrossRef][Medline] [Order article via Infotrieve]
65. Kamakura, S., Oishi, K., Yoshimatsu, T., Nakafuku, M., Masuyama, N., and Gotoh, Y. (2004) Nat. Cell Biol. 6, 547-554[CrossRef][Medline] [Order article via Infotrieve]
66. Song, M. R., and Ghosh, A. (2004) Nat. Neurosci. 7, 229-235[CrossRef][Medline] [Order article via Infotrieve]
67. Nakashima, K., Yanagisawa, M., Arakawa, H., Kimura, N., Hisatsune, T., Kawabata, M., Miyazono, K., and Taga, T. (1999) Science 284, 479-482[Abstract/Free Full Text]
68. Viti, J., Feathers, A., Phillips, J., and Lillien, L. (2003) J. Neurosci. 23, 3385-3393[Abstract/Free Full Text]
69. Joh, T., Darland, T., Samuels, M., Wu, J. X., and Adamson, E. D. (1992) Cell Growth & Differ. 3, 315-325[Abstract]
70. Bain, G., Ray, W. J., Yao, M., and Gottlieb, D. I. (1994) BioEssays 16, 343-348[CrossRef][Medline] [Order article via Infotrieve]
71. Williams, R. K., Goridis, C., and Akeson, R. (1985) J. Cell Biol. 101, 36-42[Abstract/Free Full Text]

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				Y. Komiya, N. Kurabe, K. Katagiri, M. Ogawa, A. Sugiyama, Y. Kawasaki, and F. Tashiro A Novel Binding Factor of 14-3-3{beta} Functions as a Transcriptional Repressor and Promotes Anchorage-independent Growth, Tumorigenicity, and Metastasis J. Biol. Chem., July 4, 2008; 283(27): 18753 - 18764. [Abstract] [Full Text] [PDF]

				K. Fortschegger, B. Wagner, R. Voglauer, H. Katinger, M. Sibilia, and J. Grillari Early Embryonic Lethality of Mice Lacking the Essential Protein SNEV Mol. Cell. Biol., April 15, 2007; 27(8): 3123 - 3130. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 281/11/7498    most recent M510881200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Urano, Y. Articles by Tashiro, F. Search for Related Content PubMed PubMed Citation Articles by Urano, Y. Articles by Tashiro, F. Social Bookmarking                      What's this?