Expression of human squamous cell differentiation marker, SPR1, in tracheobronchial epithelium depends on JUN and TRE motifs.

Tracheobronchial epithelial (TBE) cells that normally do not express the squamous cell differentiation marker gene, SPR1, can be induced to produce it by 12-O-tetradecanoylphorbol-13-acetate (TPA). The regulation of SPR1 gene expression by TPA occurs, in part, at the transcriptional level in primary human and monkey TBE cells. Using a transient transfection assay, we observed that TPA stimulates the activity of the reporter gene, chloramphenicol acetyltransferase, by 2-4-fold in transfected TBE cells. However, this chloramphenicol acetyltransferase activity is cell type-specific with significantly less activity in transformed epithelial cell lines and no activity in non-epithelial cell types. TPA-dependent stimulation can also be demonstrated by cotransfection with plasmid DNAs that overexpress the JUN family of proteins, especially c-JUN. Overexpression of c-JUN and TPA treatment synergistically stimulate the SPR1 promoter activity by more than 40-fold. Deletion analysis of the promoter region demonstrates that the DNA fragment of the first 98 base pairs of the 5′-flanking region contains the basal promoter activity, while the region between −162 and −96 contains the cis-enhancer elements for both the basal and TPA/c-JUN-stimulating promoter activities. This observation is supported by in vivo genomic footprinting studies that reveal persistent protections in the following motifs of this region: −141 TRE, −131 GT, −123 ETS-like, and −111 TRE-like motifs and in the enhanced protections in −141 TRE and −111 TRE-like motifs in cells after the TPA treatment. Site-directed mutagenesis in this region demonstrates the involvement of both −141 TRE and −111 TRE-like motifs in TPA/c-JUN-dependent stimulation as well as enhanced basal transcriptional activity. However, it is primarily the −111 TRE-like motif that is involved in the mediation of the enhanced basal promoter activity of the human SPR1 gene. These results are further supported by gel mobility shift assays that demonstrate the involvement of c-JUN and these TRE motifs in the formation of the DNA-protein complex.

The small proline-rich protein (SPR) 1 family with a molecular mass ranging from 10 to 30 kDa was first reported by Kartasova and van de Putte in 1988 (1). They demonstrated that the synthesis of SPR proteins is rapidly induced in human keratinocyte cultures after UV irradiation or treatment with TPA. Both of these treatments enhance the cornification of keratinocytes in culture. Two distinct groups of SPR cDNA clones were subsequently isolated using the differential hybridization technique, and their sequences were determined (1,2). Immunohistochemical studies, using a polyclonal antibody specific to the C-terminal peptides of the SPR1 protein, demonstrate the presence of a SPR1 antigen in the suprabasal cell layer of various human squamous tissues such as the epidermis and esophagus (3). A close association between the expression of SPR genes and squamous epithelial cell differentiation has been further demonstrated by Northern blot analysis (4) and in situ hybridization (5).
A unique feature of the structure of the SPR gene family is that the central segments of the encoded polypeptides are built up from tandemly repeated units of either eight (SPR1 and SPR3) or nine (SPR2) amino acids with the general consensus XKXPEPXX (6). The function of such a repeated peptide unit is currently unknown. Backendorf and Hohl (7) suggested that the SPR proteins are potential substrates involved in squamous cell cornification based on a comparison of both the Nand C-terminal amino acid sequences of SPR proteins with involucrin and loricrin, the cornified envelope proteins. Marvin et al. (8) have suggested that, based on Western blot analysis, the SPR proteins are part of the cornified envelope. We have recently demonstrated a similar finding. Unexpectedly, we also observed SPR1-like antigens in the nucleus (9). A similar observation was recently made by Hohl et al. (5) in epidermis. This finding suggests that the SPR1-like protein may also play a regulatory role in gene expression.
In contrast to squamous tissues, the presence of SPR proteins is very low in respiratory tract epithelia that normally express mucociliary functions; however, we have demonstrated a rapid increase of SPR1 gene product in isolated human and monkey airway epithelial cells upon plating on a culture dish (4). This increase can be reduced by supplementing the culture medium with vitamin A or its synthetic retinoid derivatives (4). Another study has demonstrated that vitamin A down-regulates the stability of SPR1 mRNA (10). We have also observed increased expression of SPR1 protein and mRNA in the patchy squamous cell metaplasia of monkey tracheal tissues after maintaining them under a vitamin A-free organ culture condition (data not shown). TPA, a potent squamous cell differentiation inducer, also stimulates SPR1 gene expression at the transcriptional level (11). These results establish a close relationship between the up-regulation of SPR1 gene expression and squamous airway epithelial cell differentiation.
The expression of squamous cell function in respiratory tract epithelium is a phenomenon that is frequently associated with injury. Squamous cell metaplasia has been implicated in the development of bronchogenic cancer (12,13).The nature of the induction of squamous cell differentiation in the conducting airway epithelium is still unresolved. Therefore, studies of SPR1 gene expression in non-squamous airway epithelial cells may be different from those carried out in epithelial cells that express only the differentiation of skin-like properties such as keratinization and cornification. Results obtained from studying squamous cell differentiation may provide essential understanding of the mechanism underlying the divergent pathways of cell differentiation in conducting airway epithelium (14).
We have isolated the human SPR1 genomic clone and have completed the DNA sequencing of the 5Ј-flanking region (11). The purpose of this communication is to use the transient transfection study, in vivo genomic footprinting, site-directed mutagenesis, and the gel mobility shift assay to elucidate the elements essential for both the basal, uninduced and TPAinducible promoter activities. We observed that the expression of the human SPR1 gene in conducting airway epithelium is dependent on JUN and TRE motifs located between Ϫ141 and Ϫ111 of the 5Ј-flanking region. Furthermore, the expression is cell type-specific, with a decrease in the promoter activity from primary epithelial cells to established cell lines, with no activity in the non-epithelial cell type.

MATERIALS AND METHODS
Cell Isolation and Culture Conditions-Primary TBE cells were isolated from rhesus monkey and human tracheobronchial tissues, which were obtained from the California Regional Primate Research Center and the Medical Center of the University of California at Davis, respectively. All procedures involved in the tissue procurement were approved by the University of California at Davis Animal Protocol Review Committee and the Human Subject Research Review Committee. Epithelial cell isolation and the culture conditions were carried out as described previously (15,16). Experiments were carried out between 7 and 14 days after the initiation of primary culture.
The immortalized normal human TBE cell line, BEAS-2B, subclone S, was obtained from J. F. Lechner (Lovelace Biomedical & Environmental Research Institute, Albuquerque, NM). This cell line was maintained in a serum-free hormone-supplemented medium (15,16). Other cell lines, HepG2 (ATCC HB8065, a hepatocellular carcinoma), Caco-2 (ATCC HTB37, a colon adenocarcinoma), and A172 (ATCC CRL1629, a glioblastoma), were obtained from the American Type Culture Collection (ATCC), and they were maintained in serum-supplemented culture conditions according to the supplier's data sheet.
Plasmids and Construction of CAT Reporter Constructs-Plasmid pBL-CAT2 contains the herpes simplex virus (HSV) thymidine kinase (tk) promoter in front of the CAT structural gene. Plasmid pBL-CAT3 is a promoterless construct. The expression plasmids encoding JUN B and JUN D cDNAs are pBR322-based vectors driven by a long terminal repeat promoter of the mouse sarcoma virus. The other expression plasmid encoding c-JUN cDNA was derived from pSVL vector (Pharmacia Biotech. Inc.) driven by the SV40 early promoter. The control plasmid pCH110 encodes ␤-galactosidase cDNA, which is driven by the SV40 promoter.
DNA Transfection and CAT Assays-DNA transfection was performed using a liposome technique (Lipofectin TM ) according to the procedure suggested by the manufacturer (Life Technologies, Inc.). Each culture dish at 70 -80% confluence was washed with serum-free culture medium and then transfected with 3 g of chimeric construct DNA, 1 g of pCH110 plasmid DNA, and 1 g of expression plasmid DNA when the cotransfection experiments were carried out. The pUC18 plasmid DNA was added to ensure that all the individual experiments contained the same total amount of DNA. TPA was added to transfected cultures at 10 ng/ml for 48 h before the harvesting. Cell extracts were prepared by freeze-thaw in 0.25 M Tris-HCl (pH 8.0). The protein concentration of cell extracts was determined by a modified Bradford technique (Bio-Rad). Equal amounts of protein were assayed for CAT activity by either a liquid scintillation method (11) or an enzyme-linked immunosorbent assay kit (Boehringer Mannheim) following the manufacturer's suggested protocol. The ␤-galactosidase activity, used as the internal control that normalized transfection efficiency, was determined by a ␤-galactosidase enzyme assay system (Promega).
Nuclear Extract Preparation and Gel Mobility Shift Assay-Nuclear extracts were prepared according to the method of Dignam et al. (17) from cultured cells, except that during extraction 600 mM NaCl was used instead of 400 mM NaCl. The binding was performed in a 20-l reaction volume containing 25 mM HEPES, pH 7.9, 10% (v/v) glycerol, 30 mM NaCl, 0.1 mg/ml bovine serum albumin, 1 mM dithiothreitol, 5 mM MgCl 2 , 0.5 mM EDTA, 0.5 g of salmon sperm DNA, and 50 -100 ng of poly(dI-dC). After incubation on ice for 10 min with 3-5 g of nuclear extract, 0.1-0.5 ng of end-labeled probe (ϳ20,000 cpm) was added and incubated at room temperature for 15-20 min. The DNA-protein complexes were resolved in native 4% acrylamide gels (30:1 ratio of acrylamide to bisacrylamide) in 0.5 ϫ Tris borate-EDTA buffer. In supershift analysis, nuclear extracts were mixed with 1-2 g of anti-c-JUN antibody (Santa Cruz Biotechnology, CA) and incubated on ice prior to adding the labeled DNA probe. For competition experiments, cold consensus AP1 oligonucleotide (Promega) was added to the reaction mixture prior to adding labeled probe.
In Vivo Dimethyl Sulfate (DMS) Footprinting-The method of in vivo DMS footprinting was described by Mueller and Wold (18). The primary TBE cells were cultured to 70 -80% confluence followed by incubation with or without TPA (10 ng/ml, Sigma) for 12 h. For in vivo DMS treatments, cells were treated with 0.1% DMS (Aldrich) for 2 min at room temperature. The DMS-treated cells were lysed in SDS buffer (0.5% SDS, 20 mM Tris-Cl, pH 8.0, 200 mM NaCl, and 20 mM EDTA) and then incubated with proteinase K (250 g/ml) (Sigma) at 37°C for 12 h. Genomic DNA was then isolated by phenol/chloroform extraction. As an in vitro control, protein-free genomic DNA was prepared and treated with DMS for 20 -30 s at room temperature. It was then cleaved by piperidine (Aldrich) and mapped by the ligation-mediated PCR method of genomic DNA sequencing (19).
DNA fragments were analyzed on 6% denaturing urea-polyacrylamide gels. The bands on the autoradiogram were detected after 24 -48 h of exposure. The reproducibility of the in vivo footprinting data was checked by analyzing genomic DNA samples prepared from three or more separate batches of DMS-treated cells.
Statistical Analysis-The StatView TM statistical program on the Macintosh computer was used to perform analysis of variance between different CAT reporter constructs. The Fisher PLSD (Protected Least Significant Difference) test was used to determine whether existing differences were significant at the 95% confidence level.

RESULTS
Basal Promoter Activity-Previous research based on the nuclear run-on assay and transient transfection study suggests that the treatment of primary airway epithelial cells with TPA stimulates the expression of the SPR1 gene at the transcriptional level (11). DNA sequence analysis of genomic clones revealed the following motifs: "TATA" box at Ϫ28; three TRElike sites at Ϫ49, Ϫ111, and Ϫ472; ETS at Ϫ55 and ETS-like at Ϫ123; GT motif (GGTGG) at Ϫ131; TRE site at Ϫ141; and CRE-like at Ϫ588 relative to the transcription start site in the 5Ј-flanking region of the human SPR1 gene (11). The Ϫ49 TRE-like site and Ϫ55 ETS site are merged together. To elucidate the functional roles of these putative cis-acting elements, a number of chimeric constructs containing various lengths of the 5Ј-flanking region and the CAT reporter gene were prepared (Fig. 1). The functional roles for the motifs of Ϫ141 TRE, Ϫ131 TRE-like, Ϫ55 ETS, and Ϫ49 TRE-like were further analyzed by site-directed mutagenesis. These constructs were used in transient transfection studies.
Initially, we observed no significant difference in either the basal or the TPA-induced CAT activities between 622-CAT3 and 2000-CAT3 construct transfected cells of primary TBE cultures (data not presented). Therefore, we focused the deletional analysis on the region from Ϫ622 to ϩ9. As presented in Fig. 2, the relative CAT activity in 622-CAT3 transient transfected cells without TPA treatment is 24-fold higher than the control cells transfected with the promoterless pBL-CAT3 DNA or 2-fold higher than those cells with pBL-CAT2 containing the tk promoter. The relative CAT activity in the absence of TPA treatment was the same among different chimeric constructs, except for the 162-CAT3, which was 4-fold higher (Fig. 2). This implies that the DNA sequences between Ϫ98 and ϩ9 of the 5Ј-flanking region contain sufficient information for the basal promoter activity. This elevation in the basal promoter activity observed in 162-CAT3, but not in 135-CAT3, implies that the DNA fragment between Ϫ162 and Ϫ135 contains a sequence responsible for the enhanced expression. In contrast, cells transfected with 451-CAT3 construct, which contains the 162-CAT3 DNA fragment plus the flanking region between Ϫ451 and Ϫ162, did not exhibit enhanced basal promoter activity. This implies that the region between Ϫ451 and Ϫ162 may contain a sequence that down-regulates this enhanced activity.
To further elucidate the region involved in the basal promoter activity, a 67-CAT3 chimeric construct that includes Ϫ55 ETS, Ϫ49 TRE-like, and Ϫ28 TATA was studied. As presented in Fig. 3, the relative CAT activity in cells transfected with this construct is very low but significantly higher (2-fold) than the promoterless pBL-CAT3 transfected cells; however, this activity is less than 10% of the 98-CAT3 transfection. To clarify this residual activity, site-directed mutations in both Ϫ55 ETS and Ϫ49 TRE-like sites were prepared. Single or double mutations in these sites have no effect on this residual CAT activity, which suggests that neither site is involved in this residual CAT activity.
Cell Type-specific Basal Promoter Activity-Both immunohistochemistry (3) and mRNA analyses (4) have demonstrated that the expression of the SPR1 gene is closely associated with squamous epithelial cell types. This specificity is preserved in the basal promoter analysis. As illustrated in Fig. 4, 162-CAT3 transfected primary human TBE cells exhibited the highest CAT activity as compared with the immortalized human normal TBE cell line, S clone, and other ATCC cell lines. The non-epithelial cell line A172 exhibited no CAT activity. In the case of transfection with the 98-CAT3 construct, which contains no enhanced sequence, all TBE cells, regardless of their origin, exhibited significantly lower levels of basal promoter The primary human TBE cells were transfected with various reporter constructs as indicated, and the control plasmid pCH110 encodes ␤-galactosidase cDNA. The transfected cells were treated with or without TPA (10 ng/ml) for 48 h. CAT activity in transfected cells was determined by a liquid scintillation method (11) and normalized with the ␤-galactosidase activity as described under "Materials and Methods." Bars show the standard errors of each experiment, which are based on three independent assays. An asterisk indicates a significant difference at the 95% confidence between the control (TPA untreated, light rectangle) and TPA-treated (dark rectangle) transfected cells.
activity. Transfected non-TBE cells exhibited no activity.
TPA/c-JUN Inducible Promoter Activity-The relative CAT activity in 622-CAT3 transfected cells was further enhanced 4-fold by TPA (Fig. 2). This enhancement of promoter activity by TPA is maintained in various deletion-construct transfected TBE cells, until the TRE-like motif at Ϫ111 is deleted in such constructs as 98-CAT3 and 67-CAT3. These results imply that the CRE-like motif at Ϫ588 and the two TRE-like motifs at Ϫ472 and Ϫ49 are not involved in mediating the TPA response.
We observed that TPA transiently stimulates the expression of the JUN family gene products (data not shown) in TBE cells prior to the stimulation of SPR1 gene expression. We then examined whether overexpression of JUN family proteins can enhance the CAT activity in the absence of TPA treatment. Cotransfection with one of the JUN family genes in 622-CAT3 transfected cells stimulated CAT activity 2-10-fold in the absence of TPA treatment (Fig. 5). Among the JUN family genes, c-JUN cotransfection was the most active. TPA treatment of these c-JUN cotransfected cells resulted in a 40-fold increase of CAT activity; however, TPA has no effect on the expression of JUN family proteins in cotransfected cells (data not shown). These results suggest that the SPR1 promoter is synergistically stimulated by TPA and c-JUN.
To further elucidate whether the regulatory sequences at the 5Ј-flanking region can enhance the promoter activity in heterologous constructs, several selective DNA fragments of SPR1 promoter were cloned to the pBL-CAT2 vector carrying the heterologous HSV-tk promoter (Fig. 1C). As shown in Fig. 6, in the absence of TPA and c-JUN cotransfection, the relative CAT activities in both 622/81-tk-CAT2 and 193/81-tk-CAT2 transfected cells were 13-and 11-fold higher than the pBL-CAT2 control. However, this enhanced activity cannot be seen in 622/540-tk-CAT2 transfected cells. This suggests that the enhanced activity located at the Ϫ193 and Ϫ81 5Ј-flanking region is capable of stimulating a heterologous promoter. The stimulations by TPA and c-JUN cotransfection are not as significant as in the homologous promoter system, only a 20 -40% stimulation. However, this stimulation was not seen in 622/540-tk-CAT2 transfected cells.
These results suggest that the DNA sequence located between Ϫ193 and Ϫ81 contains the cis-element that is also capable of activating the heterologous tk promoter in response to TPA/c-JUN treatment. To elucidate the site(s) responsible for this stimulation, we carried out site-direct mutations in Ϫ141 TRE and Ϫ111 TRE-like motifs. As illustrated in Fig. 7, double mutations at these two sites (Ϫ141M, Ϫ111M) significantly reduced the enhancement on tk promoter activity; however, a single mutation at Ϫ141 TRE motif (Ϫ141M) has no effect. Yet, a single mutation at the Ϫ111 TRE-like site (Ϫ111M) reduced the enhanced activity by 50%. In contrast to the enhanced tk promoter activity, a single mutation in either of these two sites has no effect on the stimulating activity exerted by the c-JUN cotransfection. These results suggest that any one of these two TRE motifs can participate in c-JUN mediated stimulation.
Genomic Footprinting of SPR1 Promoter-To further understand the nature of the regulation of SPR1 gene expression in vivo, the interactions between the promoter DNA sequence and transcriptional proteins were studied by DMS footprinting. Using appropriate primers as described in Fig. 8A, the DNAprotein interaction sites of the SPR1 promoter region were mapped. Genomic footprinting data of the DNA fragment be-tween Ϫ164 and Ϫ94 are displayed in Fig. 8B. The G residue at Ϫ140 of the coding strand of the TRE motif was partially protected, while the flanking G residue at Ϫ143 was hyperreactive in primary TBE cells. This protection at Ϫ140 G residue was slightly enhanced by TPA. In addition, a displayed protection pattern at Ϫ138 G residue was observed only in TPAtreated cells. Other protection residues in the coding strand included the G residues at Ϫ130, Ϫ127, Ϫ126, Ϫ121, Ϫ120, which are parts of the GT (Ϫ131 to Ϫ127) and ETS-like (Ϫ123 to Ϫ116) motifs. At the Ϫ111 TRE-like motif site, the G residue at Ϫ110 was partially protected, and this protection was slightly enhanced in cells after the TPA treatment. The G residues at Ϫ113, Ϫ112, and Ϫ99 in other flanking regions were partially protected, whereas the G residue at Ϫ94 was hyperreactive to piperidine cleavage in both TPA-treated or untreated cells. On the noncoding strand, only G residues at Ϫ104 and Ϫ106 displayed protections (Fig. 8B).
Other DMS footprints were detected around the CRE-like motif. On the coding strand, similar protections occurring at Ϫ587 and Ϫ606 G residues were observed in both TPA-treated and untreated cells (Fig. 9B). On the noncoding strand, G residue at Ϫ582 was partially protected, whereas G residues at Ϫ589, Ϫ593, and Ϫ598 were hyperreactive. Again, TPA had no effect on the footprinting pattern in this region.
A summary of these footprinting studies is presented in Fig.  10. There are multiple protections in the region between the Ϫ141 TRE and the Ϫ111 TRE-like motifs. These protections, presumably due to the interactions between the trans-activation proteins and the DNA sequence, are further enhanced on both Ϫ141 TRE and Ϫ111 TRE-like motifs by TPA. In contrast, FIG. 6. JUN proteins and TPA synergistically activate SPR1 promoter activity. Primary TBE cells were cotransfected with a 622-CAT3 construct in combination with c-JUN, JUN B, or JUN D expression plasmids and the control plasmid pCH110 as described under "Materials and Methods." The transfected cells were incubated with or without TPA (10 ng/ml) for 48 h prior to harvesting, and the CAT activity was determined as described in Fig. 2. The relative CAT activity was further normalized to the 622-CAT3 construct alone in transfected cells with no TPA treatment. The data were the mean of three independent dishes, and the variation was within 20%. The partial and completely filled bars represent the activities from untreated and TPAtreated cells, respectively. the protections in the CRE-like motif at Ϫ588 are not affected.
Identification of Nuclear Factors That Bind to the Ϫ162/Ϫ96 Fragment of the SPR1 Promoter-The DNA-protein interactions were further studied by using the gel mobility shift assay. A Ϫ162/Ϫ96 probe containing the TRE sites (Ϫ141 TRE and Ϫ111 TRE-like) forms a retarded complex in S-cell nuclear extracts (Fig. 11A, lane 2). The specificity of the DNA-protein complex was demonstrated by a self-competition with the unlabeled DNA probe of the region (Fig. 11A, lane 3) and by the DNA fragment known to bind the AP1 transcriptional protein complex (Fig. 11A, lanes 4 -6). Furthermore, this binding is cell type-specific. As shown in Fig. 11, incubation of the nuclear extracts isolated from fibroblasts (A172) (Fig. 11A, lane 7) with Ϫ162/Ϫ96 probe did not show any retarded complex. Sitedirected mutagenesis in either of these two TRE sites has no effect on the DNA-protein complex formation (data not shown); however, double mutations in both TRE sites (Ϫ141 and Ϫ111) result in a loss of DNA-protein complex formation (Fig. 11B). These results suggest that these two TRE sites participate in DNA-protein complex formation.
To further characterize this DNA-protein complex, anti-c-JUN antibody was used to demonstrate a supershift in this gel mobility shift assay experiment. As shown in Fig. 11C, a preincubation of S-cell nuclear extracts with the anti-c-JUN antibody significantly reduced the retarded complex and caused a supershift in gel mobility shift assay. The control experiment indicated no complex formation between the DNA probe and the antibody used in this study (Fig. 11C, lane 3). Furthermore, no gel shift was observed when the pre-immune serum was used during the preincubation period (data not shown). These results support the notion that c-JUN is involved in this DNAprotein complex formation. DISCUSSION The small proline-rich protein gene, SPR1, provides a model for identifying and characterizing the transcription factors and mechanisms that control squamous cell differentiation. In this study, we used transient transfection studies, a DMS footprinting approach, site-directed mutagenesis, and a gel mobility shift assay to characterize the 5Ј-regulatory elements of the human SPR1 gene that are responsible for the basal and TPAinducible promoter activities. We identified three distinct ele-  Fig. 8; B, data from Fig. 9. Symbols and labels used are described in Fig. 8. ments that work in concert to regulate the basal promoter activity in cells without the TPA treatment. First, we observed that the first 98-base pair DNA fragment at the 5Ј-flanking region of the human SPR1 gene contains sufficient information for the basal promoter activity. A deletion between Ϫ98 and Ϫ67 substantially reduces this activity. Neither the Ϫ55 ETS or the Ϫ49 TRE-like motif is involved in the residual activity. This is consistent with the presence of the "TATA" box at the Ϫ28 position in this DNA fragment.
Our second finding regarding the regulation of the basal promoter activity of the human SPR1 gene was that the enhanced element in the 5Ј-flanking region was identified. In the homologous promoter system, the fragment at Ϫ162 to Ϫ135 is recognized as the enhancer that elevates the basal promoter activity. However, in a heterologous promoter system such as the pBL-CAT2, which contains the HSV-tk promoter, the enhanced activity is located at Ϫ162/Ϫ96. Double mutations at the Ϫ141 TRE and Ϫ111 TRE-like motifs can eliminate this enhanced activity, suggesting the participation of both motifs in enhancing the promoter activity. The Ϫ111 TRE-like motif plays a more important role in this enhanced activity since a mutation in this site reduced the activity more than 50%. These studies are consistent with the in vivo footprinting data.
The third element in the regulation of SPR1 promoter activity is the suppresser sequence that is located at the 5Ј-flanking region between Ϫ451 and Ϫ162. The nature of this suppressive effect has not been characterized.
We also identified the regulatory elements that are involved in TPA-induced promoter activity. Several potential TREs are identified in the 5Ј-flanking region of the human SPR1 gene based on the DNA sequence information; however, not all of the potential motifs are involved. This is consistent with the report of Morrow et al. (20) that the TRE motif in the human glutathione S-transferase gene promoter is unresponsive to both TPA and the JUN/FOS protein activation. Based on the results from transient transfection studies and the DMS footprinting data, we hypothesize that the DNA fragment between Ϫ162 and Ϫ96 is involved in the mediation of TPA responsiveness. This region contains Ϫ141 TRE, Ϫ131 GT, Ϫ123 ETS-like, and Ϫ111 TRE-like motifs.
This mediation occurs not only on the homologous promoter but also, less actively, on heterologous promoters such as the HSV tk promoter. The reason for the decreased activity on the HSV tk promoter is not clear. It is possibly due to the fact that this region contains strong basal enhanced elements that can stimulate the tk promoter more than 30-fold, and further stimulation by TPA and c-JUN is restricted. Nevertheless, it is possible to demonstrate that double mutations in both Ϫ141 TRE and Ϫ111 TRE-like motifs will knock out the c-JUN-dependent stimulation. A single mutation in either site cannot eliminate this enhanced activity. These results suggest that either the Ϫ141 TRE or the Ϫ111 TRE-like site can be used for TPA/c-JUN mediated activation. This notion is further supported by the gel shift and in vivo genomic footprinting analysis. Gel shift analysis using S-cell nuclear extracts reveals that the mutation of TRE motifs (Ϫ141 and Ϫ111) significantly blocks the protein-binding complex (Fig. 11) at the Ϫ162/Ϫ96 5Ј-flanking region. Competition experiments using the consensus AP1 oligonucleotide completely abolishes the binding of nuclear factors, indicating that the Ϫ162/Ϫ96 region is probably bound by AP1 proteins. Further, preincubation of nuclear extracts with an anti-c-JUN antibody is able to supershift the DNA-protein complex (Fig. 11C). These studies indicate that TRE motifs at the 5Ј-flanking region are occupied by transcriptional factors such as AP1 proteins and c-JUN.
In vivo genomic footprinting data also revealed a persistent protection of G residues in this DNA fragment; however, the protections on the G residues in both the Ϫ141 TRE and Ϫ111 TRE-like motifs were further enhanced in cells treated with TPA. Actually, a new protection on the Ϫ138 G residue of the Ϫ141 TRE motif was induced in cells after the TPA treatment (Fig. 10). The Ϫ141 TRE (TGAGTCA) has a perfect nucleotide sequence, which matches a known consensus sequence of a TRE. While the Ϫ111 TRE-like (TGAaTCA) has A substituted FIG. 11. Gel mobility shift analysis of the ؊162/؊96 5-flanking region of SPR1 promoter. Preparation of nuclear extracts and gel mobility shift assays were performed as described under "Materials and Methods." The arrowhead in all three panels indicates the specific retarded band formed with nuclear extracts, and the asterisk in panel C indicates the supershifted band. A, A 5Ј-end-labeled double-stranded probe was incubated with 3-5 g of nuclear extracts (NE). Lane 1 represents the probe in the absence of nuclear extracts. Lane 2 represents a probe incubated with S-cell nuclear extracts. Lane 3 represents a probe incubated with nuclear extracts in the presence of an unlabeled double-stranded Ϫ162/Ϫ96 probe (self competitor, 100-fold molar excess). A consensus AP1 oligonucleotide (Promega) was included as a cold competitor at increasing molar excess with respect to the labeled probe (25-, 50-, and 100-fold molar excess in lanes 4 -6, respectively). Lane 7 represents a probe incubated with A172 (fibroblasts) nuclear extracts. B, S-cell nuclear extracts were incubated with wild type Ϫ162/Ϫ96 end-labeled fragment (lane 1), mutated (Ϫ141 and ϪTRE motifs, see Fig. 7 for details) Ϫ162/Ϫ96 labeled fragment (lane 2). C, supershift analysis. S-cell nuclear extracts were incubated with 1-2 g of anti-c-JUN antibody before adding a 5Ј-end-labeled double-stranded Ϫ162/Ϫ96 probe as described under "Materials and Methods." Lane 1, nuclear extracts without antibody; lane 2, nuclear extracts preincubated with c-JUN antibody; and lane 3, c-JUN antibody without nuclear extracts.
for G in the fourth position, the motif should retain 25-75% of the binding activity of the JUN-FOS complex (20). We have also demonstrated that this Ϫ111 TRE-like motif plays a more important role than the Ϫ141 TRE motif in mediating both the basal and stimulated enhanced activities.
However, the mediation by the Ϫ111 TRE-like motif is not that straightforward. We observed less inducible CAT activity by TPA in cells transfected with the 113-CAT3 construct despite the fact that the Ϫ111 TRE-like motif is included in this construct. In contrast, TPA responsiveness was demonstrated in cells transfected with 135-CAT3, which includes the flanking region between Ϫ135 and Ϫ111 in addition to the Ϫ111 TRElike motif. These data imply that the presence of a flanking sequence between Ϫ135 and Ϫ111 is critical for the Ϫ111 TRE-like motif to mediate the TPA responsiveness, especially in the homologous promoter system. The DNA fragment between Ϫ135 and Ϫ111 contains both GT and ETS-like motifs at Ϫ131 and Ϫ123, respectively. Interestingly, the in vivo DMS footprinting study demonstrated that the multiple protection persistently appeared on the G residues of these motifs, regardless of the TPA treatment. This result further supports the notion that the flanking region is important to the TRE-like motif in mediating TPA response. Further experiments are needed to elucidate the role of this flanking region in mediating the human SPR1 promoter activity.
We also demonstrated that c-JUN is involved in the activation of promoter activity. Cotransfection with c-JUN expression plasmid DNA can mimic the action of TPA and can also cooperate with the TPA treatment to synergistically stimulate the promoter activity. A number of TPA-inducible genes have been characterized (21)(22)(23)(24)(25)(26)(27)(28), and the TRE-like motif, consensus sequence TGA(G/C)TCA, in their promoter/enhancer region has been recognized (29 -31). This study is consistent with the notion that the TRE activity is mediated by the AP1 transcription factor, which is composed of both the JUN and the FOS family of proteins (32)(33)(34). The JUN family proteins (35)(36)(37), c-JUN, JUN B, and JUN D, can either homodimerize or heterodimerize with the FOS family proteins, c-FOS, FOS B, FRA-1, and FRA-2 (38). These dimers bind to the TRE site, thereby regulating gene expression in response to TPA (34, 39 -41). Northern blot analysis demonstrates that TPA transiently enhances the c-JUN message in primary TBE cells (data not shown). In contrast, the messages for JUN B and JUN D are very low in primary TBE cells (data not shown). Therefore, it is possible that one action of the TPA is to enhance the c-JUN synthesis. This would explain why c-JUN cotransfection also stimulates the promoter activity; however, this action is different but very complementary to the TPA treatment.
We observed a synergistic activation of the SPR1 promoter activity by both c-JUN cotransfection and the TPA treatment. This suggests that complementary pathways are activated by these treatments in the enhancement of the SPR1 promoter activity. It is necessary to point out that TPA treatment did not enhance the expression of c-JUN protein in c-JUN cotransfected cells. Therefore, the main mechanism of the TPA effect does not depend on the c-JUN production per se but rather on the activation of various kinases that further activate the c-JUN. One potential mechanism to account for the increase in c-JUN transcription factor activity would be the dephosphorylation of phosphoserine and phosphothreonine residues adjacent to the DNA binding domain of c-JUN protein. These residues are a target for casein kinase II and, when phosphorylated, inhibit the ability of c-JUN to bind to DNA (42). TPA usually activates the protein kinase C (PKC), which appears to be the regulator for c-JUN phosphatase that dephos-phorylates these residues, thereby stimulating c-JUN binding and transcriptional activation.
Lastly, we demonstrated that the basal and the enhanced promoter activities of the SPR1 gene observed in TBE cells cannot be demonstrated in other non-TBE cells. The nature of this cell type-specific mechanism is not understood; however, a similar cell type-specific and tissue-specific SPR1 gene expression has been demonstrated by immunohistochemistry (1) and Northern blot hybridization (4). This study suggests that the cell type specificity occurs at the transcriptional level. Since transcriptional regulation involves the DNA-protein interactions, it is likely that these non-TBE cells either lack transcriptional factor(s) to recognize this SPR1 DNA sequence or contain inhibitory factor(s) that interfere with the transcription of the SPR1 gene. Gel mobility shift assays using nuclear extracts from SPR1 expressing (TBE cells) and nonexpressing (nonepithelial) cell lines show formation of cell type-specific retarded complex at the SPR1 promoter region. Further detailed study may help elucidate the nature of this cell type specificity.
In summary, our results suggest the involvement of Ϫ141 TRE and Ϫ111 TRE-like motifs and c-JUN expression in conferring the maximal expression of the human SPR1 gene in TBE cells. Both Ϫ141 TRE and Ϫ111 TRE-like motifs are involved in the basal and TPA-stimulated enhanced activities. Other TRE sites, such as Ϫ472 TRE-like and Ϫ49 TRE-like, are not involved. The exact molecular basis of these motifs and the corresponding binding factors involved in the SPR1 transcription, however, remain to be established.