The small proline-rich protein (SPR) ()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 N- and 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 5`-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 TPA-inducible 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 -141 and -111 of the 5`-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

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

Various 5`-flanking regions (between -2000 and +9 relative to the transcription start site) of the human SPR1 gene were amplified from human genomic clone (11) by polymerase chain reaction (PCR) with two restriction sites attached to the two specific primers: SalI to the 5`-primer and XbaI to the 3`-primer. Positive clones containing the appropriate inserts were determined by PCR screening and confirmed by restriction mapping and DNA sequencing. The generation of various chimeric constructs is described in Fig. 1. The 2000-CAT3, 622-CAT3, 557-CAT3, 162-CAT3, 113-CAT3, 98-CAT3, and 67-CAT3 constructs consist of DNA fragments between -2000 and +9, -622 and +9, -557 and +9, -162 and +9, -113 and +9, -98 and +9, and -67 and +9, respectively, of the SPR1 promoter in the pBL-CAT3 vector under XbaI and SalI cloning sites. The chimeric constructs 622/81-tk-CAT2, 622/540-tk-CAT2, 193/81-tk-CAT2, and 162/96-tk-CAT2 contain various 5`-flanking sequences of SPR1 gene between -622 and -81, -622 and -540, -193 and -81, and -162 and -96 relative to the transcription start site, respectively, in the pBL-CAT2 vector under XbaI and SalI cloning sites.

Figure 1: Construction of various SPR1 promoter-CAT reporter constructs. A, identification of the motifs in the SPR1 5`-flanking region. The location of the 5`-end of the motif in the human SPR1 promoter is identified. Various SPR1 5`-flanking regions were PCR amplified by primers containing either a 5`-XbaI or a 3`-SalI cloning site for direct cloning into the pBL-CAT3 (B) and pBL-CAT2 (C) vectors.

The PCR reactions were carried out in a total volume of 100 µl containing 10 mM Tris-HCl, pH 8.4, 50 mM KCl, 1.2 mM MgCl, 0.01% gelatin, 250 µM each of dATP, dGTP, dCTP, and dTTP (Pharmacia). Initial denaturation was at 95 °C for 5 min followed by 30 cycles of 94 °C denaturation (1 min), 55 °C annealing (1 min), and 72 °C extension (4 min), with a final 72 °C extension for 7 min in an automated thermal cycler (Perkin-Elmer Corp.).

The 451-CAT3 and 135-CAT3 constructs containing regions between -451 to +9 and -135 to +9 of the SPR1 promoter, respectively, were constructed by ligation of SspI-XbaI and HincII-XbaI DNA fragments, digested from 622-CAT3 construct into pBL-CAT3 vector.

DNA Transfection and CAT Assays

Nuclear Extract Preparation and Gel Mobility Shift Assay

In Vivo Dimethyl Sulfate (DMS) Footprinting

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

RESULTS

Basal Promoter Activity

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 -622 to +9. 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 -98 and +9 of the 5`-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 -162 and -135 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 -451 and -162, did not exhibit enhanced basal promoter activity. This implies that the region between -451 and -162 may contain a sequence that down-regulates this enhanced activity.

Figure 2: Deletion analysis of the SPR1 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.

To further elucidate the region involved in the basal promoter activity, a 67-CAT3 chimeric construct that includes -55 ETS, -49 TRE-like, and -28 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 -55 ETS and -49 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.

Figure 3: Characterization of the 5`-flanking region of the SPR1 gene involved in basal promoter activity. Transfection of primary human TBE cells with various chimeric constructs and CAT assays were carried out as described under ``Materials and Methods.'' A, partial DNA sequences of wild type 67-CAT3 and various mutant forms. The 67-CAT3 (55M) construct is mutated at the -55 ETS site, while 67-CAT3 (49M) is mutated at the -49 TRE-like site, and 67-CAT3 (49M, 55M) has mutations in both sites. Information regarding the 98-CAT3 construct is presented in Fig. 1. B, mean and standard error of relative CAT activity (units of -galactosidase) in cells transfected with these constructs.

Cell Type-specific Basal Promoter Activity

Figure 4: Cell type specificity of basal promoter activity. Primary human and monkey TBE cells (HTBE and MTBE, respectively), and several established human cell lines as presented were transfected with 162-CAT3 and 98-CAT3 as described in the text. Data analysis was carried out similar to that described in Fig. 3. The control, pBL-CAT3 transfected human TBE cells, is included.

TPA/c-JUN Inducible Promoter Activity

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.

Figure 5: Enhancer activity of various SPR1 promoter regions and the response to TPA and c-JUN. Primary TBE cells were transfected with various reporter constructs containing 622/81-tk-CAT2, 622/540-tk-CAT2, and 193/81-tk-CAT2 constructs alone or cotransfected with c-JUN expression plasmid. The transfected cells were treated with or without TPA (10 ng/ml) for 48 h. CAT activity was determined as described in Fig. 2. The data were the mean of three independent dishes.

To further elucidate whether the regulatory sequences at the 5`-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 -193 and -81 5`-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.

Figure 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 TPA-treated cells, respectively.

These results suggest that the DNA sequence located between -193 and -81 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 -141 TRE and -111 TRE-like motifs. As illustrated in Fig. 7, double mutations at these two sites (-141M, -111M) significantly reduced the enhancement on tk promoter activity; however, a single mutation at -141 TRE motif (-141M) has no effect. Yet, a single mutation at the -111 TRE-like site (-111M) 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.

Figure 7: Identification of TRE sites involved in both basal and c-JUN inducible enhanced promoter activities. Wild type 162/96-CAT2 construct was prepared as described in Fig. 1. Constructs with mutations at -141 TRE and -111 TRE-like are shown in A. The 162/96-CAT2 (111M) construct has a mutation at -111 TRE-like, while 162/96-CAT2 (141M) is mutated at -141 TRE, and 162/96-CAT2 (111M, 141M) is a double mutant of both sites. B, relative CAT activity in transfected primary human TBE cells treated with c-JUN cotransfection (dark rectangle) or without the cotransfection (light rectangle).

Genomic Footprinting of SPR1 Promoter

Figure 8: In vivo DMS footprinting of the -193 to -81 5`-flanking region of the SPR1 promoter. A, identification of the locations and orientations of the primers used in the Sequenase and PCR amplification (indicated by arrows). B, both coding (left) and noncoding (right) strands of -193 to -81 regions were analyzed. The vertical open bars indicate the positions of different motifs. Protected and hyperreactive G residues are denoted by open and closed circles, respectively, and the numbers indicate the SPR1 promoter sequence. Sizes of the circles indicate the relative extents of DMS protection in vivo. Lane 1, control (C), protein-free genomic DNA; lane 2, genomic DNA from untreated primary TBE cells; lane 3, genomic DNA from TPA-treated primary TBE cells.

Other DMS footprints were detected around the CRE-like motif. On the coding strand, similar protections occurring at -587 and -606 G residues were observed in both TPA-treated and untreated cells (Fig. 9B). On the noncoding strand, G residue at -582 was partially protected, whereas G residues at -589, -593, and -598 were hyperreactive. Again, TPA had no effect on the footprinting pattern in this region.

Figure 9: In vivo DMS footprinting of the -622 to -540 5`-flanking region of the SPR1 promoter. A, identification of the locations and orientations of the primers used in the Sequenase and PCR amplification (indicated by arrows). B, both coding (left) and noncoding (right) strands of -622 to -540 regions were analyzed. Symbols and labels used are the same as those in Fig. 8.

A summary of these footprinting studies is presented in Fig. 10. There are multiple protections in the region between the -141 TRE and the -111 TRE-like motifs. These protections, presumably due to the interactions between the trans-activation proteins and the DNA sequence, are further enhanced on both -141 TRE and -111 TRE-like motifs by TPA. In contrast, the protections in the CRE-like motif at -588 are not affected.

Figure 10: Summary of in vivo DMS footprinting analyses of the SPR1 promoter in primary TBE cells. A, data from Fig. 8; B, data from Fig. 9. Symbols and labels used are described in Fig. 8.

Identification of Nuclear Factors That Bind to the -162/-96 Fragment of the SPR1 Promoter

Figure 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 5`-end-labeled double-stranded probe was incubated with 3-5 µg 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 -162/-96 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 -162/-96 end-labeled fragment (lane 1), mutated (-141 and -TRE motifs, see Fig. 7for details) -162/-96 labeled fragment (lane 2). C, supershift analysis. S-cell nuclear extracts were incubated with 1-2 µg of anti-c-JUN antibody before adding a 5`-end-labeled double-stranded -162/-96 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.

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 DNA-protein 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 5`-regulatory elements of the human SPR1 gene that are responsible for the basal and TPA-inducible promoter activities. We identified three distinct elements 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 5`-flanking region of the human SPR1 gene contains sufficient information for the basal promoter activity. A deletion between -98 and -67 substantially reduces this activity. Neither the -55 ETS or the -49 TRE-like motif is involved in the residual activity. This is consistent with the presence of the ``TATA'' box at the -28 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 5`-flanking region was identified. In the homologous promoter system, the fragment at -162 to -135 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 -162/-96. Double mutations at the -141 TRE and -111 TRE-like motifs can eliminate this enhanced activity, suggesting the participation of both motifs in enhancing the promoter activity. The -111 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 5`-flanking region between -451 and -162. 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 5`-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 -162 and -96 is involved in the mediation of TPA responsiveness. This region contains -141 TRE, -131 GT, -123 ETS-like, and -111 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 -141 TRE and -111 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 -141 TRE or the -111 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 (-141 and -111) significantly blocks the protein-binding complex (Fig. 11) at the -162/-96 5`-flanking region. Competition experiments using the consensus AP1 oligonucleotide completely abolishes the binding of nuclear factors, indicating that the -162/-96 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 5`-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 -141 TRE and -111 TRE-like motifs were further enhanced in cells treated with TPA. Actually, a new protection on the -138 G residue of the -141 TRE motif was induced in cells after the TPA treatment (Fig. 10). The -141 TRE (TGAGTCA) has a perfect nucleotide sequence, which matches a known consensus sequence of a TRE. While the -111 TRE-like (TGAaTCA) has A substituted 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 -111 TRE-like motif plays a more important role than the -141 TRE motif in mediating both the basal and stimulated enhanced activities.

However, the mediation by the -111 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 -111 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 -135 and -111 in addition to the -111 TRE-like motif. These data imply that the presence of a flanking sequence between -135 and -111 is critical for the -111 TRE-like motif to mediate the TPA responsiveness, especially in the homologous promoter system. The DNA fragment between -135 and -111 contains both GT and ETS-like motifs at -131 and -123, 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, 30, 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, 40, 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 dephosphorylates 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 -141 TRE and -111 TRE-like motifs and c-JUN expression in conferring the maximal expression of the human SPR1 gene in TBE cells. Both -141 TRE and -111 TRE-like motifs are involved in the basal and TPA-stimulated enhanced activities. Other TRE sites, such as -472 TRE-like and -49 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.

FOOTNOTES

*

Supported in part by National Institutes of Health Grants DE10742, HL35635, ES06230, and ES06553 and California Tobacco-related Disease Research Program Grant 3RT-0409. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

Contributed equally to this paper.

¶

Present address: Biological Dosimetry Group, Lawrence Livermore National Laboratory, Livermore, CA 94551.

**

To whom all correspondence should be addressed. Tel.: 916-752-2648; Fax: 916-752-2880.

()

The abbreviations used are: SPR, small proline-rich protein; TPA, 12-O-tetradecanoylphorbol-13-acetate; TBE, tracheobronchial epithelium; TRE, TPA-responsive element; CRE, cAMP-responsive element; tk, thymidine kinase; CAT, chloramphenicol acetyltransferase; DMS, dimethyl sulfate; GT, GGTGG motif; ETS, E-26 transformation specific; HSV, herpes simplex virus; PCR, polymerase chain reaction.

ACKNOWLEDGEMENTS

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