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Mol Cell Biol, May 1998, p. 3010-3020, Vol. 18, No. 5
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Induction of Sp1 in Differentiating Human Embryonal Carcinoma Cells Triggers Transcription of the Fibronectin Gene

Mitsuhiro Suzuki,1 Eri Oda,1 Takuma Nakajima,1 Souei Sekiya,2 and Kinichiro Oda1,*

Department of Biological Science and Technology, Science University of Tokyo, Noda 278,1 and Department of Obstetrics and Gynecology, Chiba University School of Medicine, Inobara, Chiba 280,2 Japan

Received 9 October 1997/Returned for modification 15 December 1997/Accepted 20 February 1998

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cells of the human embryonal carcinoma line NEC14 proliferate as densely packed clusters consisting of small, polygonal stem cells and do not express a detectable level of fibronectin (FN). Upon induction of differentiation by treatment with N,N'-hexamethylene bisacetamide (HMBA), the level of FN mRNA increased steeply within 24 h and FN began to be accumulated, along with the organization of actin filaments in the cells. The FN promoter elements required for the activation were analyzed in reference to a cluster of GC boxes by using the chloramphenicol acetyltransferase (CAT) gene fused to 5' sequential-deletion derivatives of the promoter and promoters carrying base substitutions in the GC boxes. Among four GC boxes, GC boxes 2 and 3 had the greatest effect on promoter activation, and base substitutions in these GC boxes resulted in 80% reduction in promoter activity. The pattern of DNA-protein complex formation with these GC boxes changed drastically after induction of differentiation. The extract prepared from undifferentiated NEC14 cells formed fast-migrating complexes (UnD complexes), while the extract prepared from NEC14 cells treated with HMBA for 24 h formed slow-migrating complexes containing Sp1. Both complexes were formed predominantly with GC box 2. Base substitutions within the GC boxes completely abolished the formation of both UnD and Sp1 complexes. Consistent with these changes, the Sp1 level increased steeply within 24 h. Induction of Sp1 expression in NEC14 cells effectively stimulated the promoter activity of the transfected FN promoter-CAT constructs. These results indicate that activation of the FN promoter in differentiating NEC14 cells occurs by the steep induction of Sp1, which prevents an undifferentiated cell factor from binding to the Sp1 sites.

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Fibronectin (FN) is a family of large glycoproteins of the extracellular matrix and has multiple domains with specific binding sites for other matrix macromolecules, such as fibrin, glycosaminoglycans, and collagen, and for its receptor, the alpha 5beta 1-type integrin on the cell surface (24). FN binds to its receptor as a dimer of similar subunits of about 250 kDa (23, 24). The cytoplasmic domain of the receptor interacts with actin filaments via several adhesion proteins, connecting the extracellular matrix to actin filaments at the receptor site called focal contacts or adhesion plaques (3, 4, 23, 25, 44). FN thus plays an important role in organizing the extracellular matrix and enabling cells to attach to it. Through these functions, FN regulates cell adhesion, migration, growth, wound healing, and tumor metastasis (12, 23).

Expression levels of FN in cells vary markedly depending on the environment and cellular capacity for proliferation. Cells transformed by various oncogenes usually express very low levels of FN (14, 17, 29), while senescent cells inevitably express high levels of FN, ceasing their proliferation (18, 35, 39). We previously showed that the level of FN gene expression in the rat derivative cell line 3Y1 transformed by adenovirus E1A and E1B genes decreased drastically to an almost undetectable level (41, 50). Analysis of regulatory factors that interact with the rat FN promoter showed that an E1A-inducible negative regulator, G10BP, binds to three G-rich sequences in the promoter (50), preventing a transcription factor, Sp1, from binding to these sites (31).

The human FN promoter (9, 10) also contains G-rich sequences, but their locations, and the number and arrangement of C residues in the G stretches, are quite different from those observed in the rat FN promoter. Reflecting the difference, purified rat G10BP does not bind to any GC boxes present in the human FN promoter. Owing to the shortage of established human cell lines that change their growth properties in response to external signals, the positive and negative transcription factors interacting with the G-rich sequences in the human FN promoter and its relation to the growth potential of cells have not yet been clarified. The human embryonal carcinoma (EC) cell line NEC14, established from a testicular germ cell tumor (46, 47), offers a good system for analysis of these factors, since it proliferates unlimitedly but can be induced to differentiate by the addition of 10-2 M N,N'-hexamethylene bisacetamide (HMBA) in vitro (20, 48). The cells tend to lose proliferative potential upon induction of differentiation, appearing larger and flattened, and finally cease their growth. Undifferentiated cells consist exclusively of stem cells that are small and polygonal, and they do not express a detectable level of FN. After induction of differentiation, however, the expression of FN increases steeply and promotes the organization of intracellular actin filaments.

In the present study, the roles of four GC boxes in the activation of the FN promoter in NEC14 cells were analyzed after induction of differentiation using 5' sequential-deletion derivatives of the promoter and promoters carrying base substitutions in these GC boxes fused to the chloramphenicol acetyltransferase (CAT) gene. The changes in the levels of regulatory factors that interact with these GC boxes were analyzed by electrophoretic mobility gel shift assays (EMSA). The results indicate that Sp1 is induced steeply shortly after the induction of differentiation, and the pattern of complex formation at these GC boxes changes drastically. Sp1 binds to all the GC boxes, although the binding affinities are different, preventing the binding of an undifferentiated cell factor (UnDF) to these GC boxes. The role of Sp1 in the induction of the FN gene and its relation to cell differentiation are discussed.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell culture. The human EC cell line NEC14, derived from testicular germ cell tumors (47), was cultivated in RPMI 1640 medium supplemented with 10% fetal calf serum. The medium was changed every day, and the cells were subcultivated by repeated pipetting at a 1:3 to 1:4 dilution when they had reached confluence. Differentiation of NEC14 cells was induced by the addition of 10-2 M HMBA (20, 48).

Construction of human FN promoter-CAT fusion plasmids. A genomic DNA clone containing the 5'-flanking region of the FN gene was isolated from a human genomic library constructed with the EMBL3 vector. The 2.8-kb insert was isolated by cleavage with EcoRI, and the fragment containing the promoter region between positions -580 and +18 was recloned into the multicloning site (MCS) of pBluescript II KS+ by using the BamHI and EcoRI linkers to generate pBShFN508 (see Fig. 2B). pBShFN508 was then cleaved with BamHI and HindIII, and the fragment containing the promoter sequence between positions -183 and +18 was inserted into the BglII-HindIII site of pSV2CAT-BX, displacing the simian virus 40 (SV40) promoter to generate phFN183CAT (see Fig. 2C). pBShFN508 was also cleaved with SacI in the MCS, and the promoter sequence was shortened by successive digestion with Exo III and mung bean nuclease as previously described (49). The DNAs were then circularized by using the BamHI linker after conversion of the cohesive termini to blunt ends. The shortened promoter sequence was isolated by cleavage with BglII and HindIII and inserted into the BglII-HindIII site of pSV2CAT-BX to generate phFN105CAT. The fusion plasmids phFN165CAT, phFN126CAT, phFN70CAT, and phFN55CAT (see Fig. 3A) were constructed from pBShFN183 by cleavage with XhoI and either AatII, EagI, Alw26I, or SmaI. The cohesive termini of the fragments containing proximal region promoter were converted to blunt ends and ligated to the BglII linkers. The fragments were then cleaved with BglII and HindIII and inserted into the BglII-HindIII site of pSV2CAT-BX. The internal-deletion derivative of the promoter used for construction of hFNDelta 183CAT was prepared from pBShFN183 by cleavage with SmaI, and the large fragment generated was circularized after removal of the small segment (positions -160 to -55). The BglII-HindIII fragment was similarly inserted into pSV2CAT-BX.

Construction of FN promoter-CAT constructs containing base substitutions in the GC boxes. Introduction of base substitutions into the GC boxes was carried out by the oligonucleotide-directed dual amber method (22). The BamHI-EcoRI fragment containing the promoter region from position -183 to +18 was isolated from pBShFN183 and cloned into the MCS of pKF19k, which carries two amber mutations within the kanamycin resistance gene. The recombinant plasmid DNA was heat denatured, and one of the following oligonucleotides carrying two to three base substitutions within the GC boxes was annealed with the selection primer containing the wild-type (WT) kanamycin resistance gene sequence to undo the amber mutations: GC box 1, 5'-GCCGGCGGGCGGGCGGGTGG-3' || GCTAGC SphI GC box 2, 5'-GGTGGGGCGGGGCGGGGACAG-3' ||| GCTAGC NheI GC box 3, 5'-CCTCCCCCGCGCCCCGGGCCT-3' ||| CCATGG NcoI GC box 4, 5'-CCAGAGGGGCGGGAGGGCCGT-3' ||| GTCGAC SalI

DNA synthesis and ligation were performed according to the manufacturer's protocol with a Mutan-Express Km kit (Takara). DNA was then transfected into Escherichia coli supE mutS cells (BMH71-18mutS) to allow replication which generates two types of plasmids, one carrying amber mutations in the kanamycin resistance gene and the other carrying base substitutions in the GC box. These plasmids were transfected into E. coli sup0 cells (MV1184), which allow replication of the latter type of plasmid only. FN promoter sequences containing base substitutions in one of the GC boxes were isolated by cleavage with BamHI and EcoRI and inserted between BamHI and EcoRI sites of pBShFN183 to generate pBShFN183mut. The pBShFN183mut DNA was cleaved with BamHI and HindIII, which cut the DNA outside the EcoRI site, and the promoter sequence from position -183 to +13 was inserted between BamHI and HindIII sites of pSV2CAT, displacing the SV40 promoter to generate phFN183CAT, which contains base substitutions in one of the GC boxes.

When base substitutions were introduced into two GC boxes, the BamHI-EcoRI fragment of pBShFN183mut was cloned into the MCS of pKF19k, and the second oligonucleotide containing the base substitutions was similarly annealed to the heat-denatured DNA. The FN promoter containing base substitutions in all the GC boxes was constructed by annealing four oligonucleotides together to the heat-denatured pKF19k DNA containing the FN promoter fragment. The base substitutions were confirmed by cleavage of the recombinant plasmid DNA with the restriction enzymes whose sites were generated by the base substitutions as shown above.

Northern blot hybridization. Northern blotting was performed with total cellular RNA prepared by the acid guanidinium thiocyanate-phenol-chloroform (AGPC) extraction method (7). RNA was subjected to electrophoresis in 1% agarose gels in buffer containing 2.2 M formaldehyde, 20 mM 3-(N-morpholino)propanesulfonic acid (MOPS) (pH 7.0), 8 mM sodium acetate, and 1 mM EDTA and was transferred to nylon membrane filters. Hybridization was carried out by adding 106 cpm of 32P-labeled DNA probe per ml. 32P labeling was carried out by the multiprime DNA labeling system (Amersham).

Western blotting. Whole-cell extracts were prepared according to the procedure of Dignam et al. (11), and 10 µg of protein was electrophoresed on 8% polyacrylamide gels with Laemmli running buffer (25 mM Tris · glycine (pH 8.3) and 0.1% sodium dodecyl sulfate) as described by Harlow and Lane (19). Proteins were electrophoretically transferred to nitrocellulose membranes (BA85; Schleicher & Schuell, Dassel, Germany) and incubated in immunoblotting diluent solution (3% gelatin, 100 ng of goat immunoglobulin G [IgG] per ml, and 0.1% Tween 20 in phosphate-buffered saline [PBS]) at room temperature for 1 h to minimize nonspecific binding of antibodies. The membrane was incubated with primary antibody at an appropriate dilution, as indicated in the figure legends, at room temperature for 1 h and was washed three times in PBS containing 0.1% Tween 20 for 15 min each time. The membrane was then incubated with secondary antibody at an appropriate dilution at room temperature for 1 h and washed three times in PBS containing 0.1% Tween 20 for 15 min each time. Immune complexes were detected by enhanced chemiluminescence (ECL) by treating the membrane with an ECL detection system according to the manufacturer's protocol (Amersham Corp.) and exposing it to X-ray film (Fuji-RX; Fuji Film, Tokyo, Japan).

EMSA. NEC14 cell extracts were prepared from nuclei by the technique of Andrews and Faller (1). The cells (5 × 106 to ~2 × 107) were washed in PBS and suspended in 800 µl of cold buffer A (10 mM HEPES-KOH [pH 7.9] at 4°C, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride). The cells were allowed to swell on ice for 10 min and were vortexed for 10 s. The cell suspension was centrifuged at 8,000 × g for 10 s at 4°C, and the pellet was resuspended in 200 to 300 µl of cold buffer C (20 mM HEPES-KOH [pH 7.9] at 4°C, 25% glycerol, 420 mM KCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride). The suspension was incubated on ice for 20 min for high-salt extraction and was centrifuged at 8,000 × g for 20 min. The supernatant was transferred to a filter cup containing a UFC3 (Millipore) filter and was concentrated about 10-fold by centrifugation at 5,000 × g for 30 min at 4°C. The concentrated supernatant was used as the cell extract after the protein concentration was adjusted to 1 µg per µl with binding buffer (20 mM HEPES [pH 7.9]-0.1 M KCl-5 mM MgCl2-2 mM EDTA-1 mM dithiothreitol-20% [vol/vol] glycerol) (38).

EMSA were performed in 20 µl of binding buffer (38) containing 1 µg of poly(dI-dC) · poly(dI-dC), 0.5 fmol (approximately 5 × 103 cpm) of 32P-labeled oligonucleotide, and extract prepared from NEC14 cells that was left untreated or treated with HMBA at 0°C for 30 min. DNA-protein complexes were resolved by electrophoresis on 5% polyacrylamide gels at 4°C for 2.5 h at 250 V in TGE buffer (25 mM Tris-hydrochloride [pH 8.0]-192 mM glycine-2 mM EDTA). For the supershift assay, the nuclear extract was preincubated with the antiserum at 0°C for 30 min prior to initiation of the binding reaction. The gels were dried and autoradiographed with an intensifying screen at -80°C.

Transient transfection and CAT assay. Subconfluent cultures of NEC14 cells were transfected with 20 µg each of FN promoter-CAT constructs by the calcium phosphate coprecipitation procedure of Chen and Okayama (5). Cell extracts were prepared after 48 h of transfection. CAT activity was assayed according to the method of Gorman et al. (15). Four hundred micrograms of protein was used for each assay. The amount of [14C]chloramphenicol converted to acetylated forms was determined by scanning the developed X-ray film with a densitometer.

Immunofluorescence. For immunofluorescence staining, NEC14 cells were seeded on a LAb-Tek Tissue Culture Chamber/Slide (Miles Scientific 4808) and allowed to adhere for 48 h. At various times after induction of differentiation, the cells on the coverslips were washed twice in cold PBS and fixed in freshly prepared aldehyde solution (4% [vol/vol] paraformaldehyde in PBS) at room temperature for 10 min. The fixed cells were washed in PBS, permeabilized in 0.2% Triton X-100 in PBS for 5 min, and washed in PBS. To minimize nonspecific binding of antibodies, the cells were preincubated in 0.2 M glycine, pH 7.4, for 1 h at room temperature. For staining of FN, the cells were covered with 500 µl of anti-FN rabbit polyclonal antibody at a dilution of 1:1,000 and were incubated at 4°C overnight. The cells were then incubated with 500 µl of fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG antibody at a dilution of 1:1,000 at 4°C overnight. For staining of actin filaments, the cells were covered with 500 µl of rhodamine-conjugated phalloidin at a dilution of 1:1,000 at 4°C overnight. In all cases, the cells were washed extensively in Tris-buffered saline and mounted in 87% glycerol (Merck) containing 2.5% 1,4-diazabicyclo[2,2,2]octane (Sigma).

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Induction of FN gene expression in NEC14 cells after induction of differentiation. The human EC cell line NEC14, established from a testicular germ cell tumor, consists exclusively of stem cells that are small and polygonal and form densely packed clusters (20, 46). The cells can be induced to differentiate in several days by the addition of 10-2 M HMBA, and the differentiated cells undergo drastic morphological changes, becoming larger and flattened, and cease to proliferate (20, 21, 48).

To show the alteration in the levels of FN, a component of the extracellular matrix, during the process of differentiation of NEC14 cells, total cellular RNAs were prepared from NEC14 cells after treatment with HMBA for varying times. The levels of mRNA were analyzed by Northern blotting. As shown in Fig. 1A, FN mRNA was scarcely detected in undifferentiated cells (0 h), but the level increased drastically after 24 h. The level decreased thereafter, and FN mRNA became scarcely detectable after 96 h. To show the correlation between the induction of FN mRNA and the change in the mRNA levels of procollagen alpha 2(I), a major component of the extracellular matrix to which FN binds, procollagen alpha 2(I) mRNA was similarly analyzed. The mRNA level of alpha 2 integrin, a subunit of the collagen alpha 2(I) receptor (43), was also analyzed, since both procollagen alpha 2(I) and alpha 2 integrin promoters have been shown to contain multiple G-rich sequences (28, 53), as does the FN promoter. As shown in Fig. 1A, the level of procollagen alpha 2(I) mRNA increased steeply within 24 h after treatment with HMBA and decreased gradually thereafter. The alpha 2 integrin mRNA level also increased steeply within 24 h, and the level was maintained throughout the differentiation process. These results suggest that the expression of these cell adhesion molecules is regulated coordinately. The level of actin mRNA was nearly constant throughout the differentiation process.


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  		FIG. 1.   Expression of FN is induced in NEC14 cells after induction of differentiation. (A) Northern blot analysis of FN, alpha 2(I) procollagen, alpha 2 integrin, and beta -actin mRNAs in differentiating NEC14 cells. NEC14 cells were treated with 10-2 M HMBA, and total cellular RNAs were prepared at the times indicated above each lane. Aliquots of 20 µg of RNA were used for Northern blotting. The EcoRI-BglII fragment (positions 5947 to 6679) of pFH1 (34), the PvuII-SacI fragment (positions 429 to 1782) of the alpha 2(I) procollagen cDNA (51), the BglII-SacI fragment (positions 2873 to 3585) of the alpha 2 integrin cDNA (53), and the EcoRI-BamHI fragment (positions 1225 to 1892) of the murine beta -actin cDNA (52) were labeled with 32P and used as probes for FN, alpha 2(I) procollagen, alpha 2 integrin, and beta -actin mRNAs, respectively. (B) Immunofluorescence staining of FN and actin filaments. NEC14 cells on coverslips were treated with 10-2 M HMBA and fixed at the times indicated. For staining of FN on the cell surface, the cells were incubated with anti-FN polyclonal antibodies and then with FITC-conjugated anti-rabbit IgG, each at a dilution of 1:1,000. Actin filaments were stained with rhodamine-conjugated phalloidin at a 1:1,000 dilution.

Expression of FN on the cell surface and the organization of actin filaments in the cytoplasm were assessed by immunofluorescence staining in order to relate the organization of the extracellular matrix of FN filaments to that of intracellular cortical cytoskeleton. NEC14 cells on coverslips were treated with HMBA, and FN was stained at the times indicated with anti-FN rabbit antibody and FITC-conjugated anti-rabbit IgG. Actin filaments in the same cells were stained with rhodamine-conjugated phalloidin, which specifically binds to actin filaments. As shown in Fig. 1B, undifferentiated NEC14 cells did not express FN on the cell surface but began to express it within 24 h, reaching a maximal level after 48 to 72 h. The level began to decrease thereafter. Very little actin filament was observed in undifferentiated NEC14 cells. Some rhodamine signal, observed as a dot, was presumably due to the appearance of spontaneously differentiated cells, which usually occurs at the peripheries of densely packed cell clusters (21). The organization of cortical actin filaments became visible after 24 h and marked at 72 h, when the cells appeared large and flattened. These results indicate that the expression of FN on the cell surface promotes the organization of intracellular cytoskeleton, as previously shown.

The promoter elements required for activation of the human FN gene in NEC14 cells following induction of differentiation. Comparison of the human FN promoter sequence between positions -240 and -30 with the corresponding rat FN promoter sequence showed the presence of multiple GC boxes in both promoters, but their locations and the base composition are significantly different. To analyze the role of these GC boxes (Fig. 2A and C) in activation of the FN promoter, 5' sequential-deletion derivatives of the promoter lacking one or more GC boxes were constructed and fused to the CAT gene (Fig. 3A). These FN promoter-CAT constructs were transfected to NEC14 cells, and CAT activities were assayed after 48 h. For estimation of the promoter activity at 24 h after treatment with HMBA, HMBA was added to the transfected cells 24 h prior to the cell harvest (Fig. 3B). The promoter activity at 72 h was estimated by transfection of NEC14 cells that were treated with HMBA for 24 h (Fig. 3B). The transfection efficiencies for the HMBA-treated and untreated NEC14 cells were not significantly different, since the promoter activity of pact-CAT, a plasmid containing the chicken beta -actin promoter fused to the CAT gene, was expressed to similar extents. The transfection was carried out twice, and average values are presented in Fig. 3A. The promoter activity of phFN183CAT was low in the undifferentiated cells but increased severalfold within 24 h. The activity was then decreased, and about 60% of the maximal activity was expressed at 72 h. This pattern of promoter activation was observed with all the CAT constructs except phFNDelta 183CAT, which carries a large internal deletion and expressed no significant activity. The promoter activities of phFN165CAT, phFN126CAT, and phFN105CAT, all of which contain GC box 2, were similar at 24 h, but the activity of phFN70CAT, which lacks GC box 2, decreased significantly, suggesting that GC box 2 plays an important role in activation of the FN promoter. GC box 3 is also important for promoter activation, since the activity of phFN55CAT, which lacks both GC boxes 2 and 3, decreased further. The activity of phFN183CAT was consistently higher than that of phFN165CAT, presumably due to the presence of the activating transcription factor-cyclic AMP response element motif in the former promoter (Fig. 2C). However, the activating transcription factor-cyclic AMP response element motif alone could not activate the promoter, since phFNDelta 183CAT showed almost no activity.


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  		FIG. 2.   A cluster of GC boxes present in the human FN promoter. (A) Nucleotide sequences of the 5'-flanking region of the human FN gene. The designation +1 marks the start site of transcription. The GC-rich sequences I, II, III, and IV used for the experiments described below are underlined. The positions of base substitutions introduced within the GC boxes are shown by the arrows. The consensus Sp1 motifs are boxed. Solid box, TATA box. (B) Structure of the recombinant plasmid pBShFN508 used for construction of 5' sequential-deletion derivatives of the human FN promoter. (C) Positions of consensus sequences for known transcription factors in the human FN promoter-CAT fusion plasmids. The positions of GC boxes 1 to 4 in the GC-rich sequences I to IV (A) are shown by the open boxes.


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  		FIG. 3.   Involvement of the GC boxes in stimulation of FN promoter activity in NEC14 cells after induction of differentiation. (A) The GC boxes present in the 5' sequential-deletion derivatives of the FN promoter in CAT constructs are shown. Numbers indicate the 5'-end positions of the promoter sequence. NEC14 cells were transfected with these CAT constructs, and CAT activities were assayed 48 h after transfection with or without treatment with HMBA. The activity expressed by phFN183CAT in undifferentiated (unD) NEC14 cells is taken as 1; the activities of other constructs are shown as relative values. The values are averages of two independent experiments with standard deviations. D, differentiated. (B) The time schedules for treatment of NEC14 cells with HMBA are shown. Bold lines represent the times when HMBA was present in the medium. TF, transfection.

To analyze the role of each GC box in FN promoter activation further, two to three base substitutions were introduced into one or more GC boxes in phFN183CAT to disrupt the Sp1 motif. To show which GC box carries base substitutions, phFN183CAT was designated phFNGGGGCAT; the four G's, from left to right, represent GC boxes 1, 2, 3, and 4. Capital G's represent WT sequences, and lowercase g's represent base-substituted sequences (Fig. 4). These human FN promoter-CAT constructs were transfected to NEC14 cells, and CAT activities were assayed after 48 h with or without treatment with HMBA. CAT activities of phFNgGGGCAT, phFNGgGGCAT, phFNGGgGCAT, and phFNGGGgCAT, each carrying base substitutions in one of the four GC boxes, were more or less reduced, but the extents of reduction were not great. A significant reduction was observed with phFNGgGGCAT, which carries substitutions in GC box 2, but it still retained more than half of the activity expressed by phFNGGGGCAT. The extents of reduction in CAT activities of the constructs carrying substitutions in two GC boxes varied considerably. Among three constructs that contained substitutions in either GC box 2, 3, or 4 in addition to GC box 1, the activity of phFNggGGCAT was most affected and that of phFNgGGgCAT was least affected, suggesting that GC box 2 plays a larger role. The larger role of GC box 2 was most evident with phFNGggGCAT, which carries substitutions in GC boxes 2 and 3. The activity was severely reduced, to less than 1/5 of that expressed by phFNGGGGCAT. GC box 3 seemed to be second in importance for promoter activity, since the activity of phFNGggGCAT was significantly lower than those of phFNggGGCAT and phFNGgGgCAT. No significant activity was expressed by phFNggggCAT, which carries base substitutions in all the GC boxes. The results obtained with the base-substituted promoter were therefore consistent with those obtained with 5' deletion derivatives of the promoter (Fig. 3) and indicate that FN promoter activity is regulated by cooperative action of these GC boxes, although GC box 2 plays a more significant role than the others.


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  		FIG. 4.   Effects of base substitutions within the GC boxes on FN promoter activity in NEC14 cells. Two to three bases within GC boxes 1, 2, 3, and 4 were substituted by the oligonucleotide-directed dual amber method (22), and phFN183CAT constructs containing base substitutions in one to four GC boxes were constructed. Here, phFN183CAT is designated phFNGGGGCAT to show the genotype of four GC boxes. The four G's, from left to right, represent GC boxes 1 through 4. Capital G's, WT sequences; lowercase g's, base-substituted sequences. The base-substituted GC boxes in the constructs are crossed out in the diagram on the left. These CAT constructs were transfected to NEC14 cells, and CAT activities were assayed 48 h after transfection with or without treatment with HMBA for 24 h. Open bars, CAT activities in undifferentiated (unD) NEC14 cells; solid bars, CAT activities in differentiation-induced (D) cells. The CAT activity expressed by phFNGGGGCAT in undifferentiated cells is taken as 1, and activities of other constructs are shown as relative values. Values are averages of two independent experiments with standard deviations.

Analyses of the DNA-protein complexes formed at the GC boxes by EMSA. To analyze a protein factor(s) that interacts with the GC boxes, the 26-bp oligonucleotides I, II, III, and IV, which contain GC boxes 1, 2, 3, and 4, respectively, and oligonucleotides of the same lengths that carry five base substitutions within the Sp1 motif, Imut, IImut, IIImut, and IVmut, were chemically synthesized as shown in Fig. 2A. DNA-protein complexes were formed with these oligonucleotides and cell extracts prepared from undifferentiated NEC14 cells (UnD extracts) and from NEC14 cells treated with HMBA for 24 and 72 h (Fig. 5). With the UnD extract, oligonucleotide II formed three fast-migrating complexes (also called UnD complexes) (Fig. 5A). The fastest-migrating complex was predominant. Oligonucleotide IV formed complexes with similar mobilities, but in much smaller amounts. No visible complex was formed with oligonucleotide I or III, but a small amount of the fastest-migrating complex was formed with increasing amounts of the extract (data not shown). A protein factor(s) associated with these complexes was termed UnDF. Introduction of base substitutions within the Sp1 motif abolished complex formation almost completely (Fig. 5B), indicating that the Sp1 sites are required for the binding of UnDF.


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  		FIG. 5.   EMSA of DNA-protein complexes formed with the GC-rich sequences in the FN promoter. (A) The DNA-protein complexes were formed with 32P-labeled 26-bp oligonucleotides I, II, III, and IV (5 fmol) shown in Fig. 2A and the extracts (4 µg of protein) prepared from undifferentiated NEC14 cells (UnD) and NEC14 cells treated with HMBA for 24 and 72 h, as indicated above each lane. Open arrow, fastest-migrating complex formed with oligonucleotide II and the UnD extract. Other arrows point to complexes that turned out to contain Sp1 or Sp3. (B) Complexes were formed as described above with the 26-bp oligonucleotides Imut, IImut, IIImut, and IVmut, each containing base substitutions within the Sp1 motif as shown in Fig. 2A. (C) Effects of anti-Sp1 and anti-Sp3 antibodies on the formation of the complexes. Complexes were formed with 32P-labeled oligonucleotide II and either the UnD or the 72-h extract in the presence of 100 ng of anti-Sp1 (PEP2) or anti-Sp3 (D20) antibodies (catalog no. sc-59X and sc-644X; Santa Cruz) as indicated above each lane. The complexes were also formed in the presence of a 500-fold molar excess of WT or base-substituted (mut) oligonucleotide II, as indicated.

With the 24-h extract, the patterns of complex formation were drastically altered (Fig. 5A). The fast-migrating complexes formed with the UnD extract were not formed at all, while a few slow-migrating complexes were formed with all the oligonucleotides. The slowest-migrating major complex, which consists of a doublet, turned out to contain the transcription factor Sp1, as described below (Fig. 5C). The complex was formed predominantly with oligonucleotide II, moderately with oligonucleotide IV, and slightly with oligonucleotides I and III. A minor complex, migrating slightly faster than the Sp1-containing complex, seemed to contain Sp3 (Fig. 5C) and was formed with oligonucleotides II and IV but not with oligonucleotides I and III. The same patterns of complex formation were observed with the 72-h extract. Oligonucleotides Imut and IVmut, which carry base substitutions in the GC boxes, formed a few complexes in much smaller quantities with all the extracts (Fig. 5B). Oligonucleotides IImut and IIImut formed no detectable complexes. The marked contrast between the WT and base-substituted oligonucleotides in the formation of the complexes suggested that activation of the FN promoter after induction of differentiation is closely related to the induction of a factor(s) which binds to these GC boxes.

Since these GC boxes contain the Sp1 motif GGGCGG, the association of Sp1 and Sp3 with the complexes was analyzed by supershift assay with the antisera raised against these factors. Sp3 is a member of the Sp1 family and acts as a bifunctional transcription factor that can both activate and repress transcription and compete with Sp1 for the Sp1 binding sites (16, 26, 36). Complexes were formed with oligonucleotide II and either the UnD or the 72-h extract in the presence of anti-Sp1 or anti-Sp3 antibody (Fig. 5C). The fast-migrating complexes formed with the UnD extract were not supershifted by these antibodies, while the slowest-migrating major complex formed with the 72-h extract was supershifted by the anti-Sp1 antibody but not by the anti-Sp3 antibody. The minor complex migrating slightly faster than the Sp1-containing complex was supershifted by the anti-Sp3 antibody, although the shift was not so clear. The formation of these complexes was completely abolished by the presence of an excess of unlabeled oligonucleotide II but not by the presence of unlabeled oligonucleotide IImut, which carries base substitutions within the Sp1 motif.

Competitive binding of Sp1 and UnDF to GC boxes. As shown in Fig. 5A, oligonucleotide II formed fast-migrating complexes with the UnD extract and slow-migrating complexes with the 24- and 72-h extracts. The formation of these complexes seemed to be mutually exclusive, since both complexes could not be formed with one type of cell extract. To analyze the competitive binding of these factors to the GC boxes, the complexes were formed with oligonucleotide II in the presence of a constant amount (4 µg of protein) of the UnD extract and increasing amounts of the 72-h extract (Fig. 6A). The amount of the fast-migrating complexes formed with the UnD extract decreased progressively along with the increase in the amount of the 72-h extract added, and in the presence of equal amounts of UnD and 72-h extracts (Fig. 6A, lane 4), the Sp1 complex was predominantly formed, abolishing the formation of the UnD complexes, indicating that these factors compete for binding to the GC box. Inversely, when the complexes were formed in the presence of a constant amount (4 µg of protein) of the 72-h extract and increasing amounts of the UnD extract, the formation of Sp1 complexes was affected only slightly (Fig. 6B). The effect of UnDF on the formation of the Sp1 complex was then compared with all the GC boxes. The complexes were formed with oligonucleotides I, II, III, and IV in the presence of 2 µg of protein of the 72-h extract and increasing amounts of the UnD extract, up to 16 µg of protein (Fig. 6C). In the absence of the UnD extract, a large amount of the Sp1 complex was formed with oligonucleotide II and a moderate amount was formed with oligonucleotide IV, but much smaller amounts were formed with oligonucleotides I and III, as observed in Fig. 5A. The formation of the Sp1 complexes with oligonucleotides I and III and the 72-h extract was abolished by the increase in the amounts of the UnD extract added, indicating that UnDF can inhibit the binding of Sp1 to GC boxes I and III when supplied in sufficient quantity. The binding of Sp1 to GC boxes 2 and 4 was also significantly inhibited by an excess of UnDF.


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  		FIG. 6.   Formation of DNA-protein complexes in the presence of both the UnD and 72-h extracts. (A and B) Competitive DNA binding of Sp1 and UnDF. (A) 32P-labeled oligonucleotide II (1 fmol) was incubated with 4 µg of protein of the UnD extract and increasing amounts of the 72-h extract, as indicated above each lane. (B) 32P-labeled oligonucleotide II was incubated with 4 µg of protein of the 72-h extract and increasing amounts of the UnD extract, as indicated. (C) 32P-labeled oligonucleotides I, II, III, and IV (1 fmol each) were incubated with 2 µg of protein of the 72-h extract and increasing amounts of the UnD extract up to 16 µg of protein, and the complexes formed were similarly analyzed. The Sp1-containing complexes are shown.

Induction of Sp1 in NEC14 cells following induction of differentiation. The results described above suggested that the activation of the FN promoter following induction of differentiation is caused by the induction of Sp1, which binds to all the GC boxes, abolishing the binding of UnDF to these GC boxes. To analyze the levels of Sp1, nuclear extracts were prepared from NEC14 cells treated with HMBA for different times. The amounts of Sp1 present in these cell extracts were analyzed by Western blotting. As shown in Fig. 7, very little Sp1 was present in the undifferentiated cells (day 0), but the level increased steeply within 24 h after induction of differentiation, consistent with the steep increase in the level of FN mRNA (Fig. 1A). The level was even higher than that in HeLa cells, which have been shown to contain abundant Sp1 (13). The amount of Sp1 present in 10 µg of protein of the 24-h extract (day 1) was roughly estimated to be 20 ng, from the density of standard purified Sp1 (5 ng) simultaneously electrophoresed. This value corresponds to 0.2% of the total proteins in the extract. The Sp1 level decreased after day 1, but a significant amount was still present on day 4 after induction of differentiation.


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  		FIG. 7.   Induction of Sp1 in NEC14 cells following induction of differentiation. NEC14 cells were treated with 10-2 M HMBA, and nuclear extracts were prepared at the times indicated above each lane. Aliquots of 5 µg of protein in the extracts were electrophoresed on a sodium dodecyl sulfate-8% polyacrylamide gel, and amounts of Sp1 were analyzed by Western blotting. Anti-Sp1 rabbit antibody was used as the primary antibody at 100 ng per ml, and goat anti-rabbit IgG conjugated with horseradish peroxidase was used as the secondary antibody at a dilution of 1:10,000. As controls, 5 ng of purified Sp1 (Promega) and 5 µg of protein of the HeLa cell extract were simultaneously assayed. The filter was treated with the ECL detection system and exposed to X-ray film for 10 s.

Stimulation of the FN promoter in undifferentiated NEC14 cells by induced expression of Sp1. To confirm the role of Sp1 in activation of the FN promoter after induction of differentiation, NEC14 cells were transfected with various FN promoter-CAT constructs with or without the Sp1 expression vector pRSV-Sp1. CAT activities were assayed 48 h after transfection. The experiment was repeated twice, and average values are presented in Fig. 8. The activities of phFN183CAT, phFN165CAT, phFN126CAT, and phFN105CAT, all of which contain GC boxes 2 and 3, cotransfected with pRSV-Sp1 increased severalfold compared with those expressed in cells transfected with these FN promoter-CAT constructs alone. The activation of phFN70CAT, which lacks GC box 2, by the Sp1 expression vector was reduced significantly, and no significant activation was observed with phFN55CAT, which lacks both GC box 2 and GC box 3. The pattern of promoter activation of various FN promoter-CAT constructs by exogenously expressed Sp1 was therefore essentially the same as that observed in the differentiation-induced cells at 24 h.


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  		FIG. 8.   Stimulation of promoter activity of 5' sequential-deletion derivatives of the FN promoter in CAT constructs by induction of Sp1 expression. Subconfluent cultures of NEC14 cells in 86-mm-diameter dishes were transfected with 15 µg each of FN promoter-CAT constructs and 5 µg of either the Sp1 expression plasmid pRSV-Sp1 (27) or pRSV0, which does not contain the CAT gene. CAT activities were assayed 48 h after transfection. The level of activity expressed by phFN183CAT cotransfected with pRSV0 was taken as 1.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The human EC cell line NEC14, established from a testicular germ cell tumor, has properties typical of EC cells (46, 47). The stem cells are small and polygonal and form densely packed clusters that do not express a detectable level of FN on their surfaces. The expression of FN in NEC14 cells increased drastically after induction of differentiation and was accompanied by the organization of actin filaments in the cytoplasm. The expression is primarily regulated at the level of transcription. The expression of FN in the extracellular spaces was shown to occur early during embryonic development, when the first major morphogenetic event involving cell migration began (41). This process may correlate with the organization of intracellular cytoskeleton.

We previously showed that repression of FN gene expression in rat 3Y1 cells by the adenovirus E1A or serum factors is primarily caused by the induction of G10BP, which binds to G-rich sequences in the promoter, preventing the binding of Sp1 to these sites (40, 41, 50). The human FN promoter also contains a cluster of G-rich sequences, although their positions and base composition are different from those in the rat FN promoter, and G10BP does not bind to any of the GC boxes in the human FN promoter. In the present studies, the GC-rich sequences in the proximal human promoter region were divided into four groups, GC boxes 1 to 4, and their roles in promoter activity were analyzed by using the CAT reporter gene fused to the 5' sequential-deletion derivatives of the promoter and promoters containing base substitutions in the GC boxes. The protein factors that bind to these GC boxes were analyzed by DNA-protein complex formation by oligonucleotides I through IV, each containing one of the GC boxes, and cell extracts prepared from undifferentiated and differentiation-induced NEC14 cells.

Transfection of NEC14 cells with various FN promoter-CAT constructs followed by induction of differentiation showed that GC boxes 2 and 3, located at positions -102 to -90 and -67 to -50, had the greatest effect on this activation, and the introduction of base substitutions in these GC boxes resulted in 80% reduction in promoter activity. The maximal activation of the FN promoter was observed 24 h after induction of differentiation, when the induction of Sp1 also became maximal. Reflecting the induction of Sp1, the pattern of DNA-protein complex formation at these GC boxes was altered markedly after induction of differentiation. The UnD extract formed fast-migrating complexes predominantly with GC box 2, weakly with GC box 4, and not at all with GC boxes 1 and 3. The formation of these UnD complexes was almost completely abolished by introduction of five base substitutions within the Sp1 motifs of the GC boxes, indicating that the UnD complexes were formed at the Sp1 site and that the binding affinity of a factor, UnDF, associating with these complexes varies drastically with the sequence surrounding the Sp1 motif. The 24- and 72-h extracts formed slow-migrating complexes but not the fast-migrating UnD complexes. The Sp1 complexes were also formed predominantly with GC box 2, moderately with GC box 4, and only slightly with GC boxes 1 and 3. This difference in the affinity of Sp1 became more evident when the amount of the 72-h extract was reduced (Fig. 6C). The order of the strengths of the binding affinity of Sp1 to the GC boxes is therefore the same as that of UnDF. The competitive binding of Sp1 and UnDF to the GC boxes was shown by complex formation in the presence of both UnD and 72-h extracts. Sp1 complex formation with GC boxes 2 and 4 was slightly affected by the presence of an excess of the UnD extract, while the formation of the Sp1 complex with GC boxes 1 and 3 was inhibited effectively by the presence of an excess of the UnD extract. Inversely, the formation of the UnD complexes with GC box 2 and the UnD extract was almost completely abolished by the presence of an equal amount of the 24- or 72-h extract. The binding affinity of Sp1 differs depending on the sequences in the GC boxes as reported previously (54), suggesting that weak Sp1 binding sites require larger amounts of Sp1. These results suggest that the steep induction of Sp1 in NEC14 cells, following induction of differentiation, drastically alters the pattern of complex formation at these GC boxes in the FN promoter, preventing the binding of UnDF.

Sp1 is a large glycoprotein with an apparent molecular mass of about 100 kDa (2). The DNA binding domain with the zinc finger motif is near the carboxyl terminus, and the transacting domain is located in the amino-terminal half and short sequences that flank the zinc fingers (8, 30, 31, 42). Three negative factors, Sp3, Sp1-I, and G10BP, that inhibit the binding of Sp1 to G-rich sequences have been reported (6, 16, 50). Sp3 has been found to be a member of the Sp1 family and has structural similarity to Sp1; however, it acts as a bifunctional transcription factor that can both activate and repress transcription by competition for the Sp1 binding sites (16, 26, 33, 36). G10BP also competes with Sp1 for binding to G-rich sequences, although its molecular mass (30 kDa) is much smaller than that of Sp1 (50). Sp1-I has a smaller molecular mass, about 20 kDa (6), and binds to both Sp1 and the retinoblastoma protein, pRB. The activation of the c-Jun promoter and the fourth promoter of the insulin-like growth factor II gene by pRB has been shown to occur through liberation of Sp1 from its inactive complex with Sp1-I (6, 32). These results suggest that there are two ways in which negative regulation of Sp1 activity occurs: one is by competitive binding to the Sp1 sites, and the other is by the formation of the complex with Sp1. UnDF, present in undifferentiated NEC14 cells, also competes with Sp1 for binding to the GC boxes. The size of UnDF seems to be much smaller than those of the members of the Sp1 family, judging from the mobility of the complexes.

The promoters of FN promoter-CAT constructs were efficiently activated in undifferentiated NEC14 cells when they were cotransfected with the Sp1 expression vector pRSV-Sp1 (Fig. 8). The pattern of activation was quite similar to that observed after induction of differentiation, suggesting that Sp1 plays a direct role in activation of the FN promoter. The involvement of Sp1 in development has been shown in mice (45). Sp1 levels are highest in developing hematopoietic cells, fetal cells, and spermatids, suggesting that an elevated Sp1 level is associated with the differentiation process. Sp1-/- embryos are retarded in development, show a broad range of abnormality, and die around day 11 of gestation, suggesting that Sp1-dependent gene activation is essential for normal development. In Sp1-/- embryos, however, the expression of many putative target genes, including cell cycle-regulated genes such as APRT, DHFR, and HPRT, was not affected, except that thymidine kinase and CpG islands, which have been shown to be the Sp1 binding sites, remained methylation free (37). The expression of the methyl-CpG-binding protein MeCP2 is reduced greatly, indicating that the MeCP2 gene is a target of Sp1. The present study on human EC cells clearly showed that the FN gene is also a target of Sp1.

Besides the FN gene, Sp1 induces the expression of the alpha 1(I) and alpha 2(I) procollagen genes (28, 51) and the alpha 2 integrin gene (53). The collagens play a crucial role in the maintenance of the structural properties of the extracellular matrix, and the integrins mediate cell attachment to the extracellular matrix by binding to FN. The simultaneous induction of the FN, alpha 2(I) procollagen, and alpha 2 integrin genes in differentiating NEC14 cells suggests that Sp1 regulates the coordinate expression of cell adhesion molecules during the differentiation process. GC boxes are the most ubiquitous promoter elements, and thus the induction of Sp1 may also stimulate transcription of other differentiation-related genes, playing a major role in the progression of cell differentiation.

    ACKNOWLEDGMENTS

We thank Yoshiaki Fujii for the Sp1 expression plasmid pRSV-Sp1, Trojanowska for the alpha 2(I) procollagen cDNA, and P. M. Pitha for the alpha 2 integrin cDNA.

    FOOTNOTES

* Corresponding author. Mailing address: Department of Biological Science and Technology, Science University of Tokyo, Yamasaki, Noda-shi, Chiba 278, Japan. Phone: 81-471-24-1501, ext. 4401. Fax: 81-471-25-1841. E-mail: koda{at}rs.noda.ac.jp.

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Abstract
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Materials & Methods
Results
Discussion
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Mol Cell Biol, May 1998, p. 3010-3020, Vol. 18, No. 5
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