CELL STRUCTURE AND FUNCTION 27: 181–188 (2002) © 2002 by Japan Society for Cell Biology
Foxa (HNF3) Up-regulates Vitronectin Expression during Retinoic Acid-induced Differentiation in Mouse Neuroblastoma Neuro2a Cells Seiko Shimizu, Mitsue Kondo, Yasunori Miyamoto∗, and Masao Hayashi Department of Biology, Ochanomizu University, Bunkyo-ku, Tokyo 112-8610, Japan
ABSTRACT. Accumulation of vitronectin protein increased in the conditioned medium of mouse neuroblastoma Neuro2a cells during retinoic acid-induced differentiation. To study the regulatory mechanism of the increase in vitronectin expression during the differentiation, the activity of the –527/+95 vitronectin promoter was observed in Neuro2a cells with or without retinoic acid treatment. The result showed that the –527/+95 promoter activity increased 2.7-fold with retinoic acid, and despite deletion of regions from –527 to –49 and +54 to +95 base pairs (bp), the –48/+53 promoter preserved the retinoic acid response. We recently showed that the –48/+53 region has two transcription factor Foxa (HNF3)-binding sites (site A from –34 to –25 bp and site B from +15 to +26 bp), suggesting that Foxa may up-regulate the vitronectin expression. Therefore, we examined the change of Foxa expression in Neuro2a cells during the differentiation. The expression of Foxa1 protein was increased during the differentiation, but the expression of Foxa2 protein was not detected. In addition, overexpression of Foxa1 increased the amount of vitronectin protein in the conditioned medium of Foxa1-overexpressed Neuro2a cells, but overexpression of Foxa2 only weakly increased it. The site-A and -B double mutation of the –527/+95 promoter remarkably reduced the promoter activity induced by Foxa overexpression, indicating that Foxa-binding sites in the –527/+95 region are located only on sites A and B. The mutation of site A in the –48/+53 promoter did not affect the retinoic acid response, but the site-B mutation abolished the constitutive promoter activity and remarkably reduced the promoter activity with retinoic acid. These results demonstrate that Foxa up-regulates the vitronectin expression during the retinoic acid-induced differentiation in Neuro2a cells.
Key words: vitronectin/Foxa1/Foxa2/neuronal differentiation/transcription
2001). In chick and mouse embryo, vitronectin is transiently expressed in the notochord, floor plate and neural tube (Seiffert et al., 1995; Martinez-Morales et al., 1997; Pons and Marti, 2000). During differentiation of granule cells in the cerebellar cortex, vitronectin is localized at the inner external germinal layer (iEGL) and internal granule layer (IGL) of the chick cerebellar cortex (Pons et al., 2001). A vitronectin receptor, integrin αvβ5, is concomitantly expressed in the granule cells, and anti-integrin αvβ5 antibody specifically inhibits the neurite extension of granule cells (Murase and Hayashi, 1998). These studies show that vitronectin and its receptor precisely express spatially and temporally in pre-neuronal cells and the surrounding cells during their neuronal differentiation. Understanding the regulatory mechanism of vitronectin expression will shed new light on the molecular mechanisms of neuronal differentiation and neuritogenesis in the motor neuron and granule cell.
Vitronectin, one of the cell adhesive proteins, regulates cell adhesion, blood coagulation, complement-mediated cell lysis and proteolytic degradation of the extracellular matrix (Tomasini and Mosher, 1991; Preissner and Seiffert, 1998; Schvartz et al., 1999). In addition, recent studies have shown that vitronectin is expressed in the neural system during embryogenesis and promotes neuronal differentiation of motor neurons (Martinez-Morales et al., 1997; Pons and Marti, 2000) and cerebellar granule cells (Pons et al., *To whom correspondence should be addressed: Yasunori Miyamoto, Ph.D., Department of Biology, Ochanomizu University, Bunkyo-ku, Tokyo 112-8610, Japan. Tel/Fax: +81–3–5978–5363 E-mail:
[email protected] Abbreviations: bp, base pairs; iEGL, inner external germinal layer; IGL, internal granule layer; FBS, fetal bovine serum; Shh, sonic hedgehog; COUP-TF I, chicken ovalbumin upstream promoter-transcriptional factor I; SDS-PAGE, SDS-polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; NEAA, non-essential amino acid; BV, basic vector.
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(Minamitani et al., 2000) except that dialysis was not performed. The protein concentration of the nuclear extracts was determined by the Bradford method (Bradford, 1976). Then, 100 µg each of nuclear protein was subjected to Western blotting.
Differentiation and neuritogenesis of neuroblastoma cells are induced in vitro by the addition of retinoic acid (Sidell, 1982; Sidell et al., 1983). Differentiating human neuroblastoma cells express vitronectin, but undifferentiated neuroblastoma cells express it only weakly or not at all (Gladson et al., 1997). We recently reported that mouse neuroblastoma Neuro2a cells express vitronectin constitutively under the control of the transcription factor Foxa (HNF3) (Shimizu et al., 2002). The vitronectin promoter has two Foxabinding sites, site A (from –34 to –25 base pairs (bp)) and site B (from +15 to +26 bp). Site B is mainly responsible for the constitutive expression of vitronectin (Shimizu et al., 2002). In this study, we examined whether the expression of vitronectin is up-regulated during retinoic acid-induced differentiation in Neuro2a cells and whether Foxa contributes to the up-regulation of vitronectin expression or not.
Site-directed mutagenesis Site-directed mutagenesis of site A (mtA) or site B (mtB) of a 101bp fragment (from −48 to +53 bp) was performed recently (Shimizu et al., 2002). In this study, site-A and -B double mutation of a 622-bp fragment (from −527 to +95 bp), which was subcloned in a plasmid, pUC119, was performed by the modified method developed by Kunkel (Kunkel, 1985; Sambrook et al., 1989). We used two synthetic oligonucleotides, which were designed recently (Shimizu et al., 2002), for mutation of site A and site B. The double-mutated fragment was digested with SmaI and SacI, and the digested fragment was inserted into the SmaI-SacI site of a Pica Gene-basic vector (Toyo Ink, Tokyo, Japan). The nucleotide sequences of the mutants were verified by a dideoxy chain termination method using [γ-32P] ATP as described (Sambrook et al., 1989). The construct, which was double-mutated at sites A and B, was designated mtA and B.
Materials and Methods Materials Mouse Foxa1 and Foxa2 cDNAs were kind gifts from Dr. Hiroshi Sasaki (Osaka University, Japan). Goat polyclonal antibodies to Foxa1 (C-20) and Foxa2 (M-20), biotin-conjugated anti-rabbit IgG and biotin-conjugated anti-goat IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Peroxidase-conjugated streptavidin was purchased from Invitrogen (Groningen, Netherlands). Anti-sera to rat vitronectin was previously prepared as described (Lee et al., 1998).
Overexpression of Foxa1 or Foxa2 in Neuro2a cells Dishes (35-mm) were precoated with 10 µg/ml fibronectin at 37°C for 1 h, and Neuro2a cells (4×105 cells per 35-mm dish) were subcultured overnight. For overexpression of Foxa1 or Foxa2, 6.9 µg of Foxa1-, Foxa2-expresson plasmid, or pcDNA1/Amp and 1.1 µg of pSV-βgal (Promega, Madison, WI USA), which is a β-galactosidase-expression plasmid for the purpose of normalizing transfection efficiency, were cotransfected into Neuro2a cells. After transfection, the cells were cultured for 12 h with MEM containing NEAA and ITS-X supplement. Then the transfected cells and conditioned media were recovered. Nuclear proteins from the transfected cells were prepared as described above, and 60 µg of each nuclear protein was subjected to Western blotting. For measurement of β-galactosidase activity, the transfected cells were lysed with a cell lysis reagent (LCβ, Toyo Ink). β-Galactosidase activity in the cell lysates was determined using o-nitrophenyl-β-Dgalactopyranoside as described (Rosenthal, 1987). The amount of each conditioned medium was normalized with each β-galactosidase activity in each transfected cell. Normalized amounts of each medium were subjected to Western blotting for vitronectin detection.
Cell culture and retinoic acid treatment Mouse neuroblastoma Neuro2a cells were grown in MEM supplemented with 10% fetal bovine serum (FBS) and non-essential amino acids (NEAA). For vitronectin detection, retinoic acid treatment was performed as follows. Dishes were precoated with 10 µg/ml fibronectin at 37°C for 1 h. Neuro2a cells (1.6×106 cells per 100mm dish for retinoic acid treatment and 1.2×106 cells per 100-mm dish for control) were subcultured on the fibronectin-coated dishes. After overnight culture, the media with or without 20 µM retinoic acid in MEM containing NEAA and ITS-X supplement (Invitrogen) were replaced. After indicated culture times, the conditioned medium was recovered. To apply the conditioned media from the same number of cells for Western blotting, we harvested the cells from the dishes and estimated the cell number from the protein concentration of the cells. The normalized amount of medium was concentrated with Ultrafree-MC (nominal molecular weight limit 5,000) (Millipore, Bedford, MA, USA), and the concentrated media were subjected to Western blotting. For Foxa1 or Foxa2 detection, Neuro2a cells (4.5×106 cells per 100-mm dish) were subcultured on three 100-mm dishes. After overnight culture, the medium was replaced with MEM containing NEAA and 2% FBS with or without 20 µM retinoic acid. After replacement of the medium, the cells were cultured for 3 h and harvested. The nuclear extracts from the harvested cells were prepared as described
Western blotting Nuclear protein or media was subjected to SDS polyacrylamide gel electrophoresis (PAGE) on 10% gels. Proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Immobilon, Millipore) for 1 h at 150 mA. Membranes were blocked in PBS containing 0.4% skim milk for Foxa1 or Foxa2 or 0.6% skim milk for vitronectin for 1 h. Then, resulting blots were probed with rabbit anti-rat vitronectin serum or goat anti-Foxa1 or goat anti-Foxa2 polyclonal antibody. The secondary antibody used was either bio-
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Results
tin-conjugated anti-goat IgG antibody or biotin-conjugated antirabbit IgG antibody. Thirdly, blots were reacted with peroxidaseconjugated streptavidin. Immunoreactive bands were detected by enhanced chemiluminescence (ECL, Amersham Biosciences, Piscataway, NJ, USA).
Accumulation of vitronectin protein is increased in the conditioned medium of Neuro2a cells during retinoic acid-induced neuronal differentiation Retinoic acid is known to induce neuronal differentiation and neuritogenesis in Neuro2a cells (Shea et al., 1985). First, we examined the vitronectin accumulation in the conditioned medium of Neuro2a cells during the retinoic acidinduced neuronal differentiation. Since serum contains vitronectin protein (Tomasini and Mosher, 1991), we performed the differentiation in a serum-free medium. After overnight culture with retinoic acid, Neuro2a cells were differentiated into neuron-like cells in serum-free medium (Fig. 1A) as well as in medium containing 2% FBS (Fig. 1B). The conditioned medium from each differentiated Neuro2a cell culture was subjected to Western blotting. The results showed that the conditioned medium from 16-h culture with retinoic acid contained a much larger amount of the vitronectin protein than the medium without retinoic acid, and that accumulated vitronectin in the conditioned medium from 20-h or 24-h culture with retinoic acid was reduced compared to that in the medium from 16-h culture (Fig. 2), indicating that vitronectin expression is up-regulated during the differentiation and suggesting that the up-regulation occurs within 16 h. Two bands were observed in the medium from 16-h culture (Fig. 2). The main band represents the intact vitronectin protein (75 kDa), and the lower band (about 50 kDa) seems to be a proteolytic fragment of the vitronectin protein. In the conditioned medium from 20h or 24-h culture, further fragmentation (less than 50 kDa) during the differentiation was observed (Fig. 2). This type of fragmentation is also observed in notochord, floor plate, neural tube and yolk (Nagano et al., 1992; Pons and Marti, 2000).
Luciferase assay Plasmid DNAs were prepared by cesium chloride density gradient as described (Sambrook et al., 1989). All transfections were repeated six times using two different preparations of DNA. One day before transfection, 4×105 cells per 35-mm dish were subcultured. The cells were transfected with 8 µg each of plasmid DNA using the calcium phosphate co-precipitation method as described (Sambrook et al., 1989). For detection of vitronectin promoter activity during the retinoic acid-induced differentiation, the plasmid DNA was a mixture of 6.9 µg of the luciferase expression construct, VN−527/+95, VN−74/+95, or VN−48/+53, which was prepared previously (Miyamoto et al., 1998), and 1.1 µg of pSV-βgal, which is a β-galactosidase expression plasmid that normalizes transfection efficiency. After transfection, the medium was replaced with MEM containing NEAA and 2% FBS with or without 20 µM retinoic acid. After 2-day culture, cells were lysed with a cell lysis reagent (LCβ, Toyo Ink). For overexpression of Foxa, 3.4 µg of the construct, VN−527/+95, site-A and -B double-mutated promoter (mtA and B) or the basic vector was cotransfected with 3.4 µg Foxa1- or Foxa2-expression plasmid, or pcDNA1/Amp and 1.2 µg pSV-βgal. After overnight culture, the transfected cells were lysed. Luciferase activity in the lysates was determined on a luminometer (Lumicounter700, Microted Co., Tokyo, Japan) according to the method of Wood (Wood, 1991). β-Galactosidase activity in the lysates was determined using o-nitrophenyl-β-D-galactopyranoside as described by Rosenthal (Rosenthal, 1987). The luciferase activity of each sample was normalized with the β-galactosidase activity. The results represent ± S.E. for six independent transfection experiments with duplicates.
Fig. 1. Retinoic acid-induced differentiation of Neuro2a cells. Neuro2a cells (1×105 cells per 35-mm dish) were subcultured with MEM containing 10% FBS. After overnight culture, the medium was replaced with serum-free medium (MEM supplemented with ITS-X) (A) or medium containing 2% FBS (B) with 20 µM retinoic acid. Further culture was performed for 1 day.
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Fig. 2. Increase of the vitronectin accumulation in the conditioned medium from Neuro2a cells during the retinoic acid-induced differentiation. Cells were subcultured on fibronectin-coated dishes overnight. The medium was then replaced with serum-free MEM with or without 20 µM retinoic acid. Each conditioned medium was recovered after 16-, 20- or 24-h culture. These samples were subjected to Western blotting using anti-vitronectin serum. To apply conditioned medium from the same number of cells for Western blotting, we harvested the cells from the dishes and estimated the cell number by the protein concentration of the cells. The normalized amount of medium was subjected to Western blotting.
–48/+53 vitronectin promoter activity increases during the retinoic acid-induced differentiation in Neuro2a cells
moter, the promoter activity of the 5’- or 3’ region, –74/+95 or –48/+53, was examined. The result showed that the both –74/+95 and −48/+53 regions retained promoter activity that was enhanced with retinoic acid (Fig. 3), indicating that the –48/+53 region has retinoic acid-responsive sites.
To examine the regulatory mechanism of the up-regulation of vitronectin expression during differentiation in Neuro2a cells, –527/+95 vitronectin promoter activity was analyzed with or without retinoic acid. The result showed that the retinoic acid-induced differentiation increased the −527/+95 promoter activity about 2.7-fold (Fig. 3). To identify the retinoic acid-responsive region in the vitronectin pro-
Up-regulation of Foxa expression during the retinoic acid-induced differentiation Because our recent study identified two Foxa-binding sites (site A from −34 to −25 and site B from +15 to +26) in this
Fig. 3. Increase of the vitronectin promoter activity during the retinoic acid-induced differentiation. A luciferase reporter construct, VN−527/+95, VN−74/ +95, or VN−48/+53 as shown on the left, was used in this luciferase assay. Neuro2a cells were transiently cotransfected with 6.9 µg of luciferase expression plasmid, VN−527/+95, VN−74/+95, or VN−48/+53 and 1.1 µg of pSV-βgal. After transfection, the medium was replaced with MEM containing 2% FBS and NEAA with or without 20 µΜ retinoic acid. After 2-day culture, the transfected cells were lysed. The activity of each transfected cell was relative to the activity of transfection with basic vector (BV) treated with 20 µΜ retinoic acid. The luciferase activity of each sample was normalized with the β-galactosidase activity. The results represent the mean ± S.E. for six independent transfection experiments with duplicate samples.
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−48/+53 region (Shimizu et al., 2002), Foxa is one of the candidates for the factor that is responsible for the up-regulation of vitronectin expression. Therefore, we examined expression of Foxa1 and Foxa2, which are members of the Foxa family, shown during neuronal differentiation (Monaghan et al., 1993). The result showed that Foxa1 was increased after 3-h culture with retinoic acid (Fig. 4), whereas expression after 6- or 8-h culture was reduced as compared to that after 3-h culture (data not shown). Foxa2 was not detected in the differentiated Neuro2a cells after culture for 3-8 h (data not shown). The neurite extension had already begun after 1-h culture, and the neurite outgrowth was proceeding after 3-h culture (data not shown). These results suggest that Foxa1 is involved in the up-regulation of vitronectin expression during the differentiation.
Overexpression of Foxa1 or Foxa2 increases the vitronectin expression Next, to examine whether Foxa overexpression is able to increase the vitronectin expression, a Foxa1 or Foxa2 expression plasmid was transfected into Neuro2a cells. The amount of Foxa1 and Foxa2 proteins expressed in the transfected cells was clearly abundant as compared to vectortransfected cells (Fig. 5A). The conditioned medium from 2-day culture of each transfected cell was subjected to Western blotting. As shown in Fig. 5B, overexpression of Foxa1 and Foxa2 increased the expression of vitronectin, and the amount of vitronectin produced with Foxa1 overexpression was larger than that with Foxa2 overexpression (Fig. 5B). These results indicate that both Foxa1 and Foxa2 are able to increase the vitronectin expression and that Foxa1 is a more prominent factor in the up-regulation of vitronectin expression than Foxa2.
Fig. 5. Induction of vitronectin expression by Foxa1 or Foxa2 overexpression. (A) Detection of the Foxa1 or Foxa2 protein using Western blotting. To detect the overexpressed Foxa1 or Foxa2 protein in Neuro2a cells after 12-h culture, transfected cells were harvested and washed with PBS. Nuclear protein was extracted from each transfected cell. Sixty µg of nuclear protein from each transfected cell was then subjected to Western blotting using anti-Foxa1 or anti-Foxa2 antibody. (B) Detection of the vitronectin expression using Western blotting. Neuro2a cells were subcultured on a fibronectin-coated dish overnight. The cells were transiently cotransfected with 6.9 µg of Foxa1- or Foxa2-expression plasmid, or pcDNA1/Amp and 1.1 µg of pSV-βgal. After transfection, the mediums were replaced with serum-free MEM. After 12-h culture, each conditioned medium was recovered. To detect the vitronectin protein, each conditioned medium was then subjected to Western blotting using anti-vitronectin serum.
Only site A and site B are Foxa-binding sites in the –527/+95 promoter region To determine whether site A and site B are the only Foxabinding sites in the –527/+95 vitronectin promoter, site-directed mutagensis of sites A and B of the −527/+95 promoter region was performed. The site-A and -B double mutation remarkably attenuated up-regulation of the promoter activity by overexpression of Foxa1 or Foxa2 (Fig. 6), indicating that sites A and B are the only Foxa-binding sites on the –527/+95 promoter. The –527/+95 promoter activity with Foxa1 overexpresson was about 3-fold higher than that with Foxa2 overexpression (Fig. 6). This result is consistent with the result of vitronectin expression induced by Foxa (Fig. 4).
Fig. 4. Increase of the Foxa1 expression during retinoic acid-induced differentiation. 4×106 cells were subcultured overnight. After subculture, the medium was replaced with MEM containing 2% FBS with or without 20 µM retinoic acid. After 3 h, the cells were harvested and washed with PBS. One hundred µg each of nuclear protein from the cells was then subjected to Western blotting using anti-Foxa1 antibody.
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Fig. 6. Effect of site-A and site-B double mutation of the −527/+95 vitronectin promoter on the increase of the promoter activity by Foxa1 or Foxa2 overexpression. To overexpress Foxa1 or Foxa2 proteins in Neuro2a cells, 3.4 µg of construct VN−527/+95, site-A and -B double-mutated promoter (mtA and B) or the basic vector was cotransfected with 3.4 µg of Foxa1- or Foxa2-expression plasmid, or pcDNA1/Amp and 1.2 µg of pSV-βgal. After overnight culture, the transfected cells were lysed. The luciferase activity and β-galactosidase activity in the lysate were measured. The activity of each transfected cell was relative to the activity of cotransfection with the basic vector (BV) and the expression vector, pcDNA1/Amp. The luciferase activity of each sample was normalized with the β-galactosidase activity. The results represent ± S.E. for six independent transfection experiments with duplicate samples.
until now. In this study, we showed up-regulation of vitronectin expression during the retinoic acid-induced neuronal differentiation in Neuro2a cells. Up-regulation of vitronectin expression is also observed in notochord and floor plate during mouse and chick neurogenesis (Seiffert et al., 1995; Martinez-Morales et al., 1997; Pons and Marti, 2000). Our previous study showed that Foxa regulates the constitutive expression of vitronectin through site B (Shimizu et al., 2002). In this study, we showed that Foxa is involved in the up-regulation of vitronectin expression during the retinoic acid-induced differentiation. Transcription factors Foxa1 and Foxa2, which are members of the Foxa family (Lai et al., 1990; Kaestner, 2000; Kaestner et al., 2000), are expressed in liver, notochord and floor plate (Monaghan et al., 1993; Sasaki and Hogan, 1993; Seiffert et al., 1995). In point of detail, the internal granular layer of the cerebellum and mouse embryonic carcinoma P19 cells express Foxa1 and/or Foxa2 during the neuronal differentiation (Jacob et al., 1997; Dahmane and Ruiz-i-Altaba, 1999). These Foxa expression patterns closely resemble the vitronectin expression pattern (Seiffert et al., 1995; Martinez-Morales et al., 1997; Adam et al., 2000; Pons and Marti, 2000; Pons et al., 2001). Our findings suggest that Foxa contributes to the upregulation of vitronectin expression in the notochord and floor plate as well as that in the retinoic acid-induced differentiated Neur2a cells. In explant culture of the chick neural
Contribution of the Foxa-binding sites, site A and site B, to the up-regulation of vitronectin expression We examined which Foxa-binding sites contribute to the up-regulation of vitronectin promoter activity. The mutation of site A did not affect the retinoic acid response (Fig. 7), indicating that site A is not involved in the increase of vitronectin promoter activity during the differentiation. However, the mutation of site B abolished the constitutive promoter activity and remarkably reduced the promoter activity with retinoic acid (Fig. 7), indicating that site B is responsible for not only the constitutive vitronectin promoter activity but also the up-regulation of vitronectin expression by retinoic acid.
Discussion Vitronectin has been reported to suppress cell proliferation and promote neuronal differentiation (Martinez-Morales et al., 1997; Pons and Marti, 2000; Pons et al., 2001). Pons et al. observed that vitronectin alters the proliferation signal from sonic hedgehog (Shh) during differentiation of granule cells and suppresses the proliferation of granule cells (Pons et al., 2001). This vitronectin function requires ingenious spatial and temporal regulation of vitronectin expression, but the mechanism of the regulation of vitronectin expression during neuronal differentiation has not been solved 186
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Fig. 7. Contribution of Foxa-binding sites in the –48/+53 promoter region to the retinoic acid response. Luciferase reporter constructs, Foxa-binding site (site A or site B)-mutated vitronectin promoter region mtA or mtB as shown on the left, were used in this luciferase assay. The mutated construct was cotransfected with pSV-βgal into Neuro2a cells. After the transfection, the medium as replaced with MEM containing 2% FBS and NEAA with or without 20 µΜ retinoic acid. After 2-day culture, the transfected cells were lysed, and the luciferase activity of each sample was normalized with the β-galactosidase activity. The activity of each transfected cell was relative to the activity of transfection with basic vector (BV) treated with 20 µΜ retinoic acid. The results represent the mean ± S.E. for six independent transfection experiments with duplicate samples.
(COUP-TFI) (Adam et al., 2000). The expression of COUPTFI has been reported to be up-regulated by retinoic acid treatment (Jonk et al., 1994). RT-PCR has shown the vitronectin mRNA level to be up-regulated by overexpression of COUP-TFI, and COUP-TFI stimulates the mouse vitronectin promoter activity in transient transfection assays (Adam et al., 2000), but it is unknown whether COUP-TFI directly binds to the vitronectin promoter region. In conclusion, expression of vitronectin is up-regulated during the retinoic acid-induced neuronal differentiation in Neuro2a cells, and it is regulated by the transcription factor Foxa. In addition, fragmented vitronectin protein has been observed in the differentiated Neuro2a cells as well as in notochord, floor plate, and neural tube during chick embryogenesis. Our findings demostrate that Foxa and vitronectin may play a role in the retinoic acid-induced differentiation of Neuro2a cells and the neuronal differentiation of motor neuron in the floor plate.
tube, it has been observed that Shh induces both vitronectin and Foxa2 expression but that vitronectin is induced earlier than that of Foxa2 (Martinez-Morales et al., 1997). This observation and our findings demonstrate that while Foxa2 is not directly involved in the vitronectin expression in Shhinduced vitronectin expression, Foxa1 may contribute to the vitronectin expression in the neural tube as well as the retinoic acid-induced differentiation in Neuro2a cells, and Foxa1 preferentially contributes to the vitronectin expression over Foxa2 during neuronal differentiation in the neural tube. Fragmentation of vitronectin, which was observed in this study, seems to be caused by proteolytic processing. A previous study observed that fragmented vitronectin is associated preferentially with Shh over the intact vitronectin molecule and that the fragmented vitronectin regulates the activity of Shh in the motor neuron differentiation of the floor plate during chick embryogenesis (Pons and Marti, 2000). Thus, we hypothesize that the fragmentated vitronectin molecule may promote neuronal differentiation in Neuro2a cells as well as motor neuron differentiation in the floor plate. Our results do not rule out the contribution of other factors to vitronectin expression during neuronal differentiation in Neuro2a cells. The up-regulation of vitronectin during the retinoic acid-induced differentiation may require additional factors. One candidate for the additional factor is chicken ovalbumin upstream promoter-transcription factor I
Acknowledgments. This study was supported by a grant for Basic Experiments Oriented to Space Station Utilization (U-36) from the Institute of Space and Astronautical Science of Japan, a grant of Ground Research Announcement for Space Utilization promoted by the Japan Space Forum, and by the Sasakawa Scientific Research Grant (14-177) from the Japan Science Society. We thank Dr. Hiroshi Sasaki for his gift of Foxa1 and Foxa2 cDNA and helpful advice, Natsumi Watanabe for her photographs of differentiated Neuro2a cells and Katherine Ono for editing the manuscript.
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