Interplay between Proximal and Distal Promoter Elements Is Required for Squamous Differentiation Marker Induction in the Bronchial Epithelium

ROLE FOR ESE-1, Sp1, AND AP-1 PROTEINS*

Sekhar P. M. Reddy § {ddagger}, Hue Vuong ¶ and Pavan Adiseshaiah ¶

From the Department of Environmental Health Sciences, The Johns Hopkins University, Baltimore, Maryland 21205, {ddagger} Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University, Baltimore, Maryland 21205

Received for publication, December 2, 2002 , and in revised form, April 4, 2003.
    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Overexpression of SPRR1B in bronchial epithelial cells is a marker for early metaplastic changes induced by various toxicants/carcinogens. Previously, we have shown that the transcriptional stimulation of SPRR1B expression by phorbol 12-myristate 13-acetate (PMA) is mainly mediated by a –150/–94 bp enhancer harboring two critical 12-O-tetradecanoylphorbol-13-acetate-responsive elements (TREs) and by Jun·Fra-1 dimers. Here, we show that a region between –54 and –39 bp containing an ETS-binding site (EBS) and a GC box is essential for both basal and PMA-inducible SPRR1B transcription. In vivo footprinting demonstrated binding of transcription factors to these elements. However, unlike enhancer TREs, exposure of cells to PMA did not significantly alter the footprinting pattern at these elements. Mutations that crippled both the EBS and GC box suppressed both basal and PMA-inducible SPRR1B transcription. Consistent with this, overexpression of EBS-binding proteins ESE-1 and ESE-3 significantly stimulated SPRR1B promoter activity. Furthermore, preceding SPRR1B transcription, PMA up-regulated mRNA expression of ETS family members such as ESE-1 and ESE-3. Although ESE-1 synergistically activated c-Jun- and PMA-enhanced SPRR1B transcription, coexpression of Sp1 and ESE-1 showed no synergistic or additive effect on promoter activity, indicating an obligatory role for AP-1 proteins in such regulation. In support of this notion, deletion or mutation of two functional TREs inhibited ESE-1- and Sp1-enhanced promoter activation. Thus, the interaction between ESE-1 and Sp1, and AP-1 proteins that bind to the proximal and distal promoter regions, respectively, play a critical role in the induction of squamous differentiation marker expression in bronchial epithelial cells.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Aberrant bronchial epithelial (BE)1 cell proliferation and differentiation following toxic injury may lead to the development of various respiratory diseases, including cancer (1, 2). BE cells, a direct target of inhaled toxicants/carcinogens, express mucous cell properties under normal conditions. However, in response to injury, they rapidly undergo changes in their structure and functions to repair the epithelium and display squamous or keratinized properties (3). This phenomenon, which was initially thought to be a protective response to form a barrier against toxicants and pollutants, may lead to epithelial cell transformation and bronchial carcinogenesis if not properly restored (1, 4, 5). Therefore, a better understanding of the molecular mechanisms controlling the squamous differentiation process will be helpful to obtain additional insight into toxicant-induced BE cell injury, repair, and carcinogenesis.

Cultured BE cells provide a good model system to delineate the molecular mechanisms governing both mucous and squamous cell functions (5). Among the genes, overexpression of human SPRR1B (small proline-rich protein 1B) is a marker for early metaplastic changes in the bronchial epithelium. SPRR1B is highly inducible in BE cells both in vitro and in vivo upon exposure to a variety of agents such as phorbol 12-myristate 13-acetate (PMA) (6, 7), carcinogens (8), and tobacco smoke (3, 9). SPRR1B belongs to a multigene family consisting of two SPRR1 genes (SPRR1A and SPRR1B), seven SPRR2 genes (SPRR2A–SPRR2F), and one each of SPRR3 and SPRR4, which display a distinct expression pattern in various epithelial cell types (10). In contrast to that in the airway epithelium, the expression of SPRR genes is abundant in various squamous tissues (8). Because of their involvement in cornification, the regulation of SPRR expression not only plays an important role in the modulation of the barrier function of various epithelia, but also provides a greater flexibility to various internal tissues (11, 12).

Previously, we demonstrated that the –150 bp SPRR1B promoter, containing two PMA- or 12-O-tetradecanoylphorbol-13-acetate-responsive elements (TREs) located at positions –139 and –109, plays a critical role in PMA-inducible SPRR1B expression in BE cells both in primary cultures and in established cell lines such as BEAS-2B clone S6 and H441 (6, 7, 13). Furthermore, we have shown that members of the AP-1 (activator protein-1) family, Jun (c-Jun, Jun-B, and Jun-D) and Fos (c-Fos, Fos-B, Fra-1, and Fra-2), differentially regulate PMA-inducible SPRR1B expression in BE cells (7). These observations indicate that an abnormal activation of AP-1 family members by toxicants plays a pivotal role in the induction of squamous cell functions in the bronchial epithelium. However, substantial experimental evidence indicates that, in addition to AP-1 proteins, the ETS transcription factor family also plays a regulatory role in epithelial cell proliferation, differentiation, and transformation. Nearly 35 ETS proteins that contain a highly conserved winged helix-turn-helix DNA-binding domain have been reported thus far (14). Upon stimulation by inducible expression, modification, and/or activation by various extracellular signals, ETS proteins bind to the core consensus sequence 5'-GGA(A/T)-3' (henceforth referred to as the ETS-binding site (EBS)) (14). Functional EBSs have been found in the promoter regions of a variety of genes that regulate cell growth and differentiation, apoptosis, angiogenesis, and inflammation (14). Some of the regulatory regions of extracellular matrix proteins and epidermal differentiation markers, including SPRR proteins, also contain EBSs in the vicinity of functional TREs (15). Several studies have shown that interaction of ETS transcription factors with other proteins such as AP-1 and Sp1 plays an important role in the regulation of keratinocyte differentiation (1519).

In this study, we have investigated whether epithelium-specific ETS subfamily members ESE-1, ESE-2, and ESE-3 and Sp1 have a similar role in the regulation of SPRR1B expression in BE cells. Here, using in vivo footprinting and mutational analysis, we demonstrate that a region between –54 and –39 bp containing the EBS and GC box participates in the regulation of both basal and PMA-inducible SPRR1B transcription. Mutation of these sites strongly suppressed both basal and PMA-inducible promoter activities. Overexpression of ESE-1, ESE-3, and Sp1 strongly stimulated SPRR1B transcription. Deletion of distal enhancer-containing TREs strongly suppressed such activation. Our results suggest that interplay between protein factors that bind at distal TREs and the proximal EBS and GC box is required for high level toxicant-inducible SPRR1B transcription in BE cells.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Gene Expression Analyses—Primary cultured BE cells were isolated from human tracheobronchial tissues and cultured as described previously (6, 7). The immortalized normal human BE cell line BEAS-2B subclone S6 (obtained from Dr. J. Yankaskas) was maintained in serum-free hormone-supplemented medium. The NCI-H441 cell line (Clara-like bronchiolar pulmonary adenocarcinoma) was maintained in RPMI 1640 medium supplemented with 5% serum according to the American Type Culture Collection data sheet. Cells were treated with either vehicle (Me2SO) or PMA (5 ng/ml for primary cultures, 20 ng/ml for H441 cells, and 100 ng/ml for S6 cells) for the indicated time periods. Total RNA (20 µg/lane) was isolated, and reverse transcription (RT)-PCR was performed as previously described (7, 13). Briefly, total RNA (750 ng) was reverse-transcribed into cDNA, and PCR was performed with an aliquot of cDNA using gene-specific primer pairs (Table I). The number of cycles employed to amplify the product was in a linear range in each case (7, 13).


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TABLE I
Oligonucleotides used for RT-PCR analysis

 

Transient Transfections Assays—The wild-type expression vectors were kindly provided by the indicated people: ESE-1 and ESE-3 cloned into pcDNA3 (Antonio Tugores, University of California, San Diego, CA), Sp1 and Sp3 cloned into pcDNA3 (Guntram Suske, Institut fur Molekularbiologie und Tumorforschung, Philipps-Universitat Marburg, Marburg, Germany), and c-Jun cloned into the pCMV vector (Michael Birrer, NCI, National Institutes of Health). Deletion and site-directed mutation of –622/+12 and –150/+12 bp SPRR1B promoter fragments were generated by PCR using specific primers and cloned upstream of the luciferase gene (Table II) as described previously (7, 13). Cells were grown in 48-well plates to 70–80% confluence and transfected with 100 ng of promoter-reporter construct, 20 –50 ng of cytomegalovirus-{beta}-galactosidase DNA, and 0 –200 ng of empty or specific expression vector using FuGENE 6 transfection agent. The luciferase activity of individual samples was normalized against {beta}-galactosidase activity and/or total protein as described previously (7, 13). Triplicate samples were analyzed for luciferase activity, and all experiments were repeated at least two to three times. Data are expressed as the mean ± S.E.


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TABLE II
Oligonucleotides used for EMSA and site-directed mutational analysis

 

In Vivo Footprinting—In vivo dimethyl sulfate (DMS) footprinting was carried out as previously described (6, 7). Briefly, cells were cultured to 70–80% confluence, followed by incubation with or without PMA for 12 h. Cells were subsequently treated with 0.1% DMS for 2 min at room temperature under respective culture conditions without serum. Genomic DNA was then isolated using the phenol/chloroform extraction method. As an in vitro control, protein-free genomic DNA was prepared and treated with DMS for 20 –30 s at room temperature. Both in vivo and in vitro treated genomic DNAs (3 µg) were subjected to piperidine treatment, and the cleaved DNA fragments were amplified by the ligation-mediated PCR method of genomic DNA sequencing (20). Amplified DNA fragments were analyzed on a 6% denaturing urea-polyacrylamide gel. Bands were detected following 24–48 h of autoradiography. DNA samples prepared from at least two separate batches of DMS-treated cells were analyzed to demonstrate the reproducibility of data.

Electrophoretic Mobility Shift Assay (EMSA)—Nuclear extracts from vehicle (Me2SO)or PMA-treated S6 cells were prepared, and EMSA was performed as described previously (7, 13). Briefly, to determine GC box-binding proteins, 0.5 ng of 32P-end-labeled SPRR1B promoter fragments (wild-type or mutants; for sequences, see Table II) or double-stranded Sp1 consensus oligonucleotide was incubated with the nuclear extracts in buffer containing 5 mM HEPES (pH 7.9), 25 mM NaCl, 10% glycerol, 500 µM EDTA, 500 µM ZnCl2, 0.2 mM phenylmethylsulfonyl fluoride, and 0.5 µg of salmon sperm DNA. To determine ETS protein binding, nuclear extracts were incubated with the indicated labeled probes in 20 µl of buffer containing 20 mM Tris-Cl (pH 7.9), 50 mM NaCl, 20% glycerol, 0.2 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, and 0.2 mg/ml bovine serum albumin (15). For supershift analysis, nuclear extracts were mixed with 1–2 µg of antibody specific for ETS-1/2 (sc-112X), Sp1 (sc-59G), or ESE-1 (sc-17306X) (all obtained from Santa Cruz Biotechnology, Santa Cruz, CA) and incubated on ice for 1–2 h prior to adding the labeled DNA probe.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In Vivo Footprinting of the SPRR1B Proximal Promoter— Previously, using in vivo footprinting and site-directed mutagenesis, we demonstrated that a 57-bp fragment (–150 to –94 bp) containing two TREs at positions –139 and –109 (Fig. 1A) is essential for PMA-stimulated expression of SPRR1B in BE cells (6, 7). Because other studies have shown that the ETS and Sp/Krüppel-like factor family of transcription factors regulates differentiation marker expression in keratinocytes and esophageal cells (1519), the present study was undertaken to examine the role of an EBS and an Sp1-like motif (henceforth referred as the GC box) located between positions –54 and –39 in the regulation of SPRR1B expression in BE cells. Importantly, the sequences and positions of these sites are highly conserved among various SPRR family members (15). The protein-DNA interactions at both coding (–71 to –29 bp) and noncoding (–69 to –18 bp) strands of the promoter (Fig. 1) were mapped at single nucleotide resolution by a genomic sequencing method in intact living cells that were untreated or PMA-treated. Compared with protein-free genomic DNA controls, both primary cultures of tracheobronchial epithelial (PTBE) cells (Fig. 1B) and the established nonmalignant BE cell line S6 (Fig. 1C) displayed strong in vivo footprints covering the –71/–29 bp SPRR1B promoter. On the coding strand, –36, –41, –42, and –45 G residues of the GC box displayed strong protection in both primary cultures and S6 cells compared with the respective naked DNA controls. On the noncoding strand, –39 and –44 G residues of the GC box also displayed protection. The –52 G residue of the EBS was protected (~50%) compared with the naked DNA control (N) in both PTBE and S6 cells. However, there was no significant alteration of the reactivity of the –52 G residue upon PMA stimulation. Other protected G residues include the –56, –62, –63, and –66 residues flanking the EBS on the 5'-end. This region also contains a GATA-like sequence. Although the overall foot-printing pattern of the promoter is similar, protection in S6 cells was somewhat weak in comparison with the pattern observed in PTBE cells. This is consistent with the nature of a high level expression of SPRR1B in PTBE cells compared with transformed nonmalignant BE cells S6 and HBE-1 (6, 7).These results indicate that the EBS and GC box located in the proximal promoter are occupied in vivo by protein factors that might play a regulatory role in SPRR1B expression in BE cells.



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FIG. 1.
In vivo DMS footprinting of the SPRR1B proximal promoter. A, DNA sequence of the –150/+38 bp 5'-flanking region of human SPRR1B. The main functional cis-elements are boxed. The TATA box is underlined. Arrows indicate the location and orientation of the primers used for in vivo footprinting (for more details, see Ref. 6). B, in vivo footprints within and flanking the EBS and GC box of the top or coding (left panel) and bottom or noncoding (right panel) strands of the proximal promoter in PTBE cells. Vertical bars on the left indicate the positions of cis-elements. Numbers on the left and right indicate the positions of G residues relative to the transcriptional start site. Protected G residues are denoted by circles. Changes in the DMS reactivities of certain G residues detected in both untreated and PMA-treated cells were quantified by densitometry scanning of the autoradiographs. The black circles represent completely protected G residues, and the striped circles represent residues that exhibit >40% protection. The white circles represent G residues that exhibit 20–40% protection. Lanes 1 and 4, protein-free DNA treated with DMS in vitro (N); lanes 2 and 5, DNA from vehicle (Me2SO)-treated PTBE cells; lanes 3 and 6, DNA from PMA-treated PTBE cells. C, in vivo footprinting in S6 cells. Lanes 7 and 10, genomic DNA treated with DMS in vitro (N); lanes 8 and 11, DNA from untreated S6 cells; lanes 9 and 12, DNA from PMA-treated S6 cells. D, summary of the in vivo footprinting within and flanking the EBS and GC box in BE cells. Only the DMS reactivities of the G residues observed in both primary and S6 cells are shown. cont, control.

 

The EBS and GC Box Regulate Both Basal and PMA-stimulated SPRR1B Transcription—To address the functional relevance of the in vivo footprints, we mutated the promoter spanning the EBS and GC box by a two-step PCR method in the context of the –620/+ 12 (referred to as 620-Luc) and –150/+ 12 (referred to as 150-Luc) bp 5'-flanking regions (Fig. 2A). Both wild-type and mutant promoter-reporter constructs (as depicted in Fig. 2A) were transiently transfected into S6 and H441 cells. We have chosen these two cell lines because both endogenous SPRR1B mRNA and promoter activity are strongly induced upon PMA treatment (6, 7, 13). The luciferase activity of the –150 bp promoter-reporter construct (150-Luc) was considered as 1. The reporter gene expression observed in the presence or absence of PMA is shown in Fig. 2B. Consistent with our previous studies (6, 7, 13), both the 620-Luc and 150-Luc constructs displayed a higher promoter activity that could be significantly induced upon PMA stimulation. However, mutations that crippled both the EBS and GC box (620EBS/GC-mt and 150EBS/GC-mt) strongly ablated the basal and PMA-inducible promoter activities, which were nearly comparable to that of the mutant lacking the TREs (620TRE-mt). Similar results were obtained in PTBE cells (data not shown). Thus, both in vivo footprinting and mutation studies suggest that the EBS and GC box located at –54 and –48 bp play a prominent role in SPRR1B transcription in BE cells.



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FIG. 2.
The –54/–39 bp proximal promoter regulates SPRR1B transcription in BE cells. A, positions of the enhancer TREs, the proximal promoter EBS, and GC box. Mutations were selectively introduced either in TREs or in the EBS/GC box by PCR as described under "Materials and Methods." B, effect of mutations on promoter activity. S6 (left panel) and H441 (right panel) cells were grown to 70–80% confluence and then transfected with 100 ng of wild-type (wt) or mutant promoter-reporter constructs along with 20 ng of {beta}-galactosidase vector. Transient transfections and luciferase (Luc) assays were carried out as described under "Materials and Methods." cont, control.

 

Overexpression of ESE-1 and Sp1 Up-regulates SPRR1B Transcription—To determine the role of the EBS and GC box, we selectively introduced mutations in the context of the –150 bp promoter (Fig. 3A) that contain necessary elements for PMA-inducible SPRR1B transcription in BE cells (6, 7, 13). Cells were transfected with wild-type or mutant promoter constructs and treated with or without PMA as described under "Materials and Methods." The mutation that crippled the EBS significantly suppressed both basal and PMA-inducible promoter activities (Fig. 3B). Interestingly, the mutation that crippled the GC box did not have a significant effect on basal activity, whereas it had some effect on PMA-inducible expression. To determine whether the EBS and GC box are required for inducible expression, cells were transfected with wild-type or mutant promoter constructs along with expression vectors coding for ESE-1, ESE-3, Sp1, and Sp3, and luciferase activity was quantified. As shown in Fig. 3C, overexpression of ESE-1, ESE-3 (data not shown), and Sp1 strongly enhanced wild-type –150 bp promoter activity nearly 5-fold, whereas Sp3 did not have any significant effect. In contrast, the mutant promoter construct lacking both the EBS and GC box (EBS/GC-mt) did not respond to ESE-1 or Sp1. More importantly, even the basal promoter activity was decreased in this construct. Selective mutation of the EBS (EBS-mt) or GC box (GC-mt) also had a similar effect on inducible reporter gene expression. Although individual mutations of both the EBS and GC did not completely eliminate ESE-1- and Sp1-inducible expression, the inducible levels were nearly comparable to that of the basal promoter activity of the wild-type construct. Thus, these studies indicate the requirement of both the EBS and GC box for high level ESE-1-, Sp1-, and PMA-inducible SPRR1B transcription in BE cells.



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FIG. 3.
Both the EBS and GC box are necessary for maximal inducible expression. To determine whether both the EBS and GC box are required for PMA-stimulated SPRR1B expression, either the EBS (EBS-mt) or GC box (GC-mt) was mutated by PCR (A), and their effects on promoter activity were analyzed (B). Transient transfections and luciferase assays were carried out as described in the legend to Fig. 2. p < 0.05 (*) and p < 0.05 (**) compared with vehicle- and PMA-treated cells transfected with the wild-type (WT) promoter, respectively. To examine the role of EBS- and GC box-binding proteins in gene regulation, SPRR1B promoter-reporter constructs with mutations in either the EBS (EBS-mt) or GC box (GC-mt) or in both (EBS/GC-mt) were transiently transfected into S6 cells along with equal amounts of parental empty, ESE-1, ESE-3 (data not shown), Sp1, and Sp3 expression vectors (C). After overnight incubation, the medium was replaced and incubated further for ~14 h. The cellular lysate was prepared, and luciferase expression was analyzed. The promoter activity of the –150 bp wild-type (WT) construct was taken as 1. Bars show the mean ± S.E. of relative luciferase activity.

 

PMA Up-regulates the Expression of ETS Transcription Factor Subfamily Members ESE-1 and ESE-3 in BE Cells—Previous studies have shown an involvement of the ETS transcription factor subfamily members ESE-1 (21), ESE-2 (22), and ESE-3 (23, 24) in the regulation of differentiation marker expression in various epithelial cell types (1519). We examined the expression pattern of ESE family members in S6 and H441 cells. Cells were treated with either vehicle or PMA for different time periods as indicated. Total RNA was isolated, and mRNAs were amplified using RT-PCR with gene-specific primers (Table I). As shown in Fig. 4A, the mRNA expression of ESE-1 was low in unstimulated S6 cells. However, PMA significantly enhanced the message levels (lane 2) as early as 90 min, and they peaked at 6 h and remained elevated through 14 h. Most importantly, PMA-inducible ESE-1 message levels preceded SPRR1B induction. The mRNA levels of ESE-2 and ESE-3 were undetectable in S6 cells. As shown in Fig. 4B, H441 cells displayed basal expression of ESE-1 (lane 1), and PMA up-regulated its mRNA in a biphasic manner. PMA induced the expression of ESE-1 as early as 30 min, reaching peak values at ~60 min. However, the expression was somewhat suppressed at 3 and 6 h and slightly elevated thereafter. Similar to S6 cells, the mRNA expression of ESE-2 was neither detectable nor inducible upon PMA stimulation. ESE-3 expression was very low or undetectable in unstimulated cells, but PMA significantly induced the message levels as early as 30 min, and they remained elevated through 3 h. At 6 h, PMA-inducible expression decreased significantly, but remained elevated thereafter at 14 h. We also examined the expression patterns of the ETS family members ETS-1 and ETS-2, which have been shown to interact with AP-1 proteins and to regulate gene expression (14). PMA treatment did not alter the basal expression of ETS-1 and ETS-2 in both S6 and H441 cells. It is noteworthy that a higher level of PCR amplification (35 cycles) was used for detection of ETS-1 and ETS-2 message levels compared with Jurkat cells (22 cycles), in which ETS-1 and ETS-2 expression was shown to be abundant (25). Both up-regulation of ESE-1 and ESE-3 message levels preceding SPRR1B transcription and stimulation of SPRR1B promoter-driven reporter gene expression by overexpression of ESE-1 and ESE-3 suggest a potential role for these transcription factors in the induction of squamous differentiation in the bronchial epithelium.



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FIG. 4.
PMA distinctly up-regulates ESE subfamily member expression in BE cells. S6 (left panel) and H441 (right panel) cells were treated with vehicle or PMA for the indicated periods, and total RNA was isolated. RT-PCR was performed using gene-specific primers (Table I) as described under "Materials and Methods."

 

Distal TREs Are Necessary for ESE-1- and Sp1-inducible SPRR1B Transcription—We next determined whether distal TREs, which play a critical role in both basal and inducible SPRR1B regulation in BE cells, are required for ESE-1- and Sp1-enhanced promoter activation. SPRR1B promoter-reporter constructs lacking TREs (Fig. 5A) were transiently transfected along with ESE-1 and ESE-3 (Fig. 5B) and Sp1 (Fig. 5C) expression vectors into S6 cells, and promoter activity was quantified. Overexpression of ESE-1 strongly up-regulated reporter gene expression driven by the –150 bp SPRR1B promoter. Although the –111 bp promoter, which lacks the –139 bp TRE but contains the –109 bp TRE, displayed an ~50% reduction in basal promoter activity, overexpression of ESE-1 strongly induced promoter activity. In contrast, a promoter-reporter construct driven by the –67 bp promoter (67-Luc), which lacks both TREs, displayed very low level basal promoter activity. Notably, overexpression of ESE-1 or Sp1 did not significantly induce promoter activity above the basal level. Together, these results indicate the importance of both TREs for high level ESE-1- and Sp1-inducible expression, whereas at least one TRE is probably sufficient for such activation.



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FIG. 5.
Distal TREs are required for ESE-1- and Sp1-enhanced SPRR1B promoter activity. Upon reaching 70–80% confluency, S6 cells were cotransfected with 100 ng of the indicated SPRR1B promoter constructs (150-Luc, 111-Luc, and 67-Luc) as shown in A and the {beta}-galactosidase vector along with 100 ng of empty, ESE-1, or ESE-3 expression vector (B) or Sp1 expression vector (C). pGL3 is a control luciferase vector driven by the SV40 minimal promoter. After overnight incubation, the medium was replaced and incubated further for ~14 h. Promoter activity assays of various samples were carried out as described in the legend to Fig. 2. cont, control.

 

ESE-1 Synergistically Transactivates c-Jun- and PMA-enhanced SPRR1B Transcription—Previously, we have shown that Jun proteins positively up-regulate SPRR1B transcription both in S6 and H441 cells (6, 7, 13). Therefore, we examined the role of ESE-1 in PMA- and c-Jun-inducible SPRR1B expression in S6 cells. As shown in Fig. 6A, overexpression of ESE-1 strongly enhanced SPRR1B promoter activity. ESE-1-in-ducible promoter activity was nearly comparable to or higher than that seen upon PMA stimulation (Fig. 6B). Moreover, overexpression of ESE-1 had an additive effect on PMA-inducible SPRR1B transcription (Fig. 6B). To examine the effect of ESE-1 on c-Jun-enhanced SPRR1B promoter activation, S6 cells were transiently transfected with the 150-Luc promoter construct along with 10 ng of c-jun and/or ESE-1 expression vector, and luciferase activity was analyzed. In this case, a minimal amount of expression vector was chosen to keep the inducible levels of reporter expression at low levels. As shown in Fig. 6C, whereas cotransfection of the wild-type c-jun expression vector enhanced promoter activity, a comparable amount of ESE-1 (10 ng) did not have an effect. However, cotransfection of c-jun (10 ng) and ESE-1 (10 ng) markedly enhanced promoter activity compared with either vector alone. Moreover, promoter activity was further enhanced in response to PMA. Together, these results indicate that ESE-1 and c-Jun cooperatively interact to synergistically stimulate SPRR1B transcription in S6 cells. Although ESE-3 up-regulates SPRR1B transcription, we did not study its role because RT-PCR studies did not reveal any detectable message levels in S6 cells treated with or without PMA.



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FIG. 6.
ESE-1 synergistically transactivates c-Jun-enhanced SPRR1B promoter-driven transcription. A, S6 cells were cotransfected with the 150-Luc promoter-reporter and {beta}-galactosidase vector along with 10 –100 ng of ESE-1 expression vector. The parental vector (empty) was added to keep the total amount of transfected DNA equal in all samples. B, cells were cotransfected with the 150-Luc promoter-reporter and {beta}-galactosidase vector along with 50 ng of ESE-1 expression vector. Cells were treated with PMA, and luciferase activity was analyzed as described in the legend to Fig. 2. C, cells were cotransfected with the 150-Luc promoter-reporter and {beta}-galactosidase vector along with empty (first and second bars), c-Jun (third and fourth bars), ESE-1 (fifth and sixth bars), or ESE-1 and c-Jun (seventh and eighth bars) expression vectors (10 ng each). Parental empty vectors were added to keep the total amount of transfected DNA equal in all samples. Luciferase activity was analyzed as described in the legend to Fig. 2. cont, control.

 

Sp1 and ESE-1 Do Not Synergistically Transactivate the SPRR1B Promoter—In vivo footprinting data (Fig. 1) demonstrated strong protein-DNA interactions at the GC box of the SPRR1B proximal promoter. Moreover, overexpression of Sp1, but not Sp3 (Fig. 3C), up-regulated promoter activity nearly 5-fold. To further define the role of Sp1 in the regulation of SPRR1B, S6 cells were transiently transfected with the 150-Luc promoter construct along with variable amounts of an Sp1 expression vector and subsequently treated with vehicle or PMA. Overexpression of Sp1 up-regulated promoter activity in a dose-dependent manner (Fig. 7A). Furthermore, Sp1 positively up-regulated PMA-inducible promoter activity in a manner similar to that observed with ESE-1. We next examined the role of Sp1 in c-Jun- and/or ESE-inducible promoter activation. Cells were transfected with the 150-Luc promoter construct along with the c-Jun (10 ng), ESE-1 (50 ng), and/or Sp1 (50 ng) expression plasmid as indicated, and reporter gene expression in unstimulated and PMA-stimulated cells was analyzed (Fig. 7B). Consistent with our previous studies (7, 13), overexpression of c-Jun activated the SPRR1B promoter, whereas coexpression of Sp1 has either a synergistic or an additive effect. Furthermore, the combined expression of ESE-1 and Sp1 did not exhibit either an additive or a synergistic effect. However, promoter activity was substantially elevated in the presence of PMA. When all three genes (ESE-1, Sp1, and c-jun) were cotransfected, basal activity was markedly elevated compared with ESE-1/Sp1- or empty vector-transfected cells. PMA-inducible promoter activity was nearly comparable to that in ESE-1/Sp1-transfected PMA-treated cells. Intriguingly, SPRR1B promoter activity in cells cotransfected with all three expression plasmids was slightly reduced to that in c-Jun- and ESE-1-transfected or c-Jun- and Sp1-transfected cells. One likely possibility for this discrepancy is that, when overexpressed, these transcription factors may titrate out/squelch certain unidentified factors such as coactivators that may be required for high level SPRR1B expression. Alternatively, it is possible that, when overexpressed, Sp1 and ESE-1 may compete to bind to their respective adjacent sites (the EBS and GC box) in the proximal promoter, thereby affecting SPRR1B transcription. Indeed, coexpression of c-Jun and ESE-1 significantly stimulated the transcription of the –150 bp promoter lacking the GC box (150GC-mt) with or without PMA in comparison with the wild-type promoter (Fig. 7C). This is consistent with EMSA results showing that the removal of EBS enhanced Sp1 binding in a PMA-dependent manner. Conversely, mutagenesis of the Sp1-binding site (GC box) strongly enhanced ETS binding (see below). These results suggest that the relative ratio and distribution of Sp1 and ESE-1 proteins may also play a role in SPRR1B transcription. A further study is needed to resolve these possibilities.



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FIG. 7.
Sp1 does not synergistically transactivate c-Jun-enhanced SPRR1B promoter driven transcription. A, S6 cells were cotransfected with the 150-Luc promoter-reporter and {beta}-galactosidase vector along with indicated amounts (expressed in nanograms) of Sp1 cDNA expression vector. B, cells were cotransfected with the 150-Luc promoter-reporter and {beta}-galactosidase vector along with the empty, c-Jun (10 ng), Sp1 (50 ng), and/or ESE-1 (50 ng) expression vector as indicated. Parental empty vectors were added to keep the total amount of transfected DNA equal in all samples. The luciferase activity of unstimulated and PMA-stimulated cells was analyzed as described in the legend to Fig. 2. C, cells were cotransfected with the wild-type (WT) or mutant (GC-mt, bearing a mutation in the GC box) 150-Luc promoter-reporter and {beta}-galactosidase vector along with c-Jun (10 ng) and ESE-1 (50 ng) expression vectors as indicated. The luciferase activity of unstimulated and PMA-stimulated cells was analyzed as described in the legend to Fig. 2. cont, control.

 

Transcription Factor Binding to the SPRR1B Proximal Promoter Elements—The above data indicate that the –54/–39 bp promoter containing the EBS and GC box is necessary for both basal and PMA-inducible SPRR1B transcription. We therefore examined the protein binding to this region by EMSAs using the wild-type and mutant (bearing mutations in the EBS and/or GC box) fragments of the –60/–36 bp SPRR1B promoter (Fig. 8A). As shown in Fig. 8B, vehicle-treated S6 nuclear extracts (lane 1) revealed the formation of multiprotein complexes I–IV at the promoter that were enhanced significantly after PMA treatment (lane 2). Mutations within both the EBS and GC box (EBS/GC-mt) strongly reduced the binding of complexes I, II, and VI, without having an effect on complex III (lanes 3 and 4). A specific mutation within the EBS markedly reduced the intensity of complexes I and II (see also Fig. 9C, lane 12), without having an appreciable effect on complex III (Fig. 8B, lanes 5 and 6). Interestingly, a mutation within the EBS markedly enhanced the band intensity of complex IV (compare lanes 1 and 5 with lanes 2 and 6). On the other hand, a mutation in the GC box completely eliminated complex VI and also diminished the intensity of complex II (lanes 7 and 8). In contrast to the EBS, a mutation within the GC box not only enhanced the intensity of protein complex I, but also had an effect on the band mobility. The specificity of complexes was also analyzed by EMSA with a 50-fold excess of the unlabeled wild-type fragment and DNA fragments of the –60/–36 bp promoter with mutations in the EBS and GC box. Preincubation with the wild-type oligonucleotide totally competed out the formation of all complexes, whereas the oligonucleotide bearing specific mutations within the EBS and GC box competed out complexes I, II, and IV (data not shown).



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FIG. 8.
Analysis of protein binding to the SPRR1B proximal promoter. Preparation of nuclear extracts and EMSA were performed as described under "Materials and Methods." The closed arrowheads indicate the positions of complexes I–IV (specific retarded bands formed with nuclear extracts), whereas the open arrowhead indicates the position of the free probe. A, shown is a schematic presentation of the wild-type (WT) and mutant fragments of the –60/–36 bp region of the SPRR1B promoter. The wild-type fragment represents the intact –60/–36 bp region containing the functional EBS and GC box. EBS/GC-mt is the –60/–36 bp promoter bearing mutations within the EBS and GC box. EBS-mt is the –60/–36 bp promoter bearing mutations within the EBS. GC-mt is the –60/–36 bp promoter bearing mutations within the GC box. For sequences, see Table II. B, nuclear extracts isolated from vehicle-treated (–) or PMA-treated (+) cells were incubated with 32P-end-labeled wild-type or mutant SPRR1B promoter fragments as indicated, and protein·DNA complexes were separated as described under "Materials and Methods."

 


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FIG. 9.
Sp1 and ESE-1 bind to the proximal promoter of SPRR1B. A, 32P-end-labeled double-stranded Sp1 oligonucleotide (obtained from Santa Cruz Biotechnology) was incubated with 2 µg of nuclear extract isolated from vehicle-treated (–; lane 2) or PMA-treated (+; lane 3) S6 cells. In lane 3, the nuclear extract isolated from PMA-treated cells was incubated in the presence of 1–2 µg of anti-Sp1 antibody ({alpha}-Sp1). F represents the free probe. The asterisk indicates the position of the supershifted complex. B, protein binding was analyzed as described for A, except that the –60/–36 bp SPRR1B promoter was used instead of the Sp1 oligonucleotide. C, S6 cell nuclear extracts were preincubated either with a 50-fold molar excess of unlabeled double-stranded SPRR1B proximal promoter (self) or with a consensus EBS. D, shown are the results from supershift analysis of nuclear extracts isolated from vehicle-treated (lanes 13–15) and PMA-treated (lanes 16 –18) cells using preimmune (Ig), ETS-1/2-specific, and ESE-1-specific antibodies. comp, competition; cont, control.

 

To further characterize the protein binding, EMSA was performed using antibodies specific for Sp1, ETS-1/2, and ESE-1. Nuclear extracts were incubated with anti-Sp1 antibody prior to addition of the labeled Sp1 consensus oligonucleotide probe (Fig. 9A) or –60/–36 bp SPRR1B promoter (Fig. 9B). PMA treatment did not change the protein complex formation at the Sp1 consensus sequence. However, anti-Sp1 antibody completely blocked and/or caused a supershift of the protein binding to the sequence. At the SPRR1B proximal promoter, PMA treatment enhanced protein·DNA complex formation (lane 7) compared with vehicle-treated controls (lane 6). Furthermore, incubation of nuclear extracts with anti-Sp1 antibody blocked formation of most of the protein·DNA complexes. Similarly, we next determined the binding of the ETS family proteins to the –60/–36 bp SPRR1B promoter. As shown in Fig. 9C, preincubation of nuclear extracts with an excess amount (50-fold molar excess over the respective labeled probe) of unlabeled SPRR1B promoter (self; lane 9) or double-stranded EBS consensus oligonucleotide (EBS; lane 10) probe blocked protein·DNA complex I formation. As shown in Fig. 9D, anti-ESE-1 antibody blocked complex I formation both in untreated (lane 15) and PMA-treated (lane 18) nuclear extracts, whereas preimmune serum and antibody specific for ETS-1/2 had no effect, indicating binding of ESE-1 to the SPRR1B promoter. Together, these results indicate an important role for ESE-1 and Sp1 transcription factor binding to the proximal promoter in SPRR1B promoter regulation.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In this study, we have shown that the proximal promoter containing the EBS and GC box located at –54 and –48 bp, respectively, is necessary for both basal and PMA-inducible expression of SPRR1B in BE cells. However, these two motifs are insufficient for gene induction in the absence of distal TREs located at positions –139 and –109. Together, these observations demonstrate a cooperative interaction between binding factors at the distal TREs and proximal cis-elements, the EBS and GC box. The EBS binds members of the ETS family of transcription factors. Among ETS proteins, overexpression of ESE-1 and ESE-3 strongly enhanced promoter activity in a dose-dependent manner, whereas ETS-1 and ETS-2 showed little activation (data not shown). Consistent with this, PMA markedly increased the expression of ESE-1 in S6 cells and ESE-1 and ESE-3 mRNA expression in Clara-like H441 cells. Although PMA induced SPRR1B expression in both cell types, the significance of a biphasic induction of ESE-1 and ESE-3 expression in H441 cells is unclear at present. It is possible that, unlike S6 cells, H441 cells express surfactants and CC-10 proteins, and a biphasic induction of ESE proteins might play a role in cellular differentiation. For example, previous studies have shown that ESE-3 regulates the expression of glandular epithelium-specific genes such as c-met and the proline-rich protein MP-6 (24). Nonetheless, the facts that PMA induces the expression of SPRR1B in both S6 and H441 cells and that S6 cells lack any detectable ESE-3 mRNA expression indicate a prominent role for ESE-1 in the regulation of SPRR1B transcription in BE cells. Consistent with this view, overexpression of ESE-1 up-regulates the expression of late differentiation markers such as SPRR1A (15), SPRR2A (16), and transglutaminase-3 (26), which participate in the formation of the cornified cell envelope, a terminal phenotype of various squamous epithelia (12). In contrast, ESE-1 down-regulates the transcription of early differentiation markers such as keratin-4 in esophageal and cervical squamous epithelial cells (17). ESE-1 is predominantly expressed in simple and stratified epithelia of various tissues, including the bronchial epithelium (21, 22). Moreover, ESE-1 is not expressed in fibroblasts and lymphocytes, in which SPRR expression is neither detectable nor inducible by PMA (data not shown). Together, these observations indicate a potential role for ESE-1 in the regulation of gene expression involved in squamous differentiation in various epithelial cell types, including the bronchial epithelium.

Paradoxically, ESE-1 (also know as ESX, Jen, ELF3, and ERT) is overexpressed in both lung large cell carcinoma and adenocarcinoma tissues as well as in several lung and non-lung malignant cell lines (27). However, we (7) and others (28) have shown very low or undetectable levels of SPRR1B in several malignant BE cell lines. For example, the A549 lung adenocarcinoma cell line, in which both basal and PMA-inducible SPRR1B mRNA levels are undetectable (7), expresses high basal levels of ESE-1 compared with S6 cells (data not shown). ESE-1 is also overexpressed in breast cancer cells (29, 30), which apparently express low levels of SPRR1B mRNA (~100-fold less) compared with normal mammary tissue (31). Thus, these studies indicate that, although ESE-1 is necessary, some other factor(s) are also required for SPRR1B expression. Indeed, we have recently shown that AP-1 proteins differentially regulate SPRR1B expression in BE cells (7, 13). Thus, it is conceivable that the expression and activation as well as the composition of transcription factors such as AP-1 and ESE-1 that bind to regulatory regions play a critical role in the induction of airway squamous cell metaplasia.

The Sp family members Sp1–Sp4 preferentially bind to GC boxes with a general consensus sequence 5'-GGGGCGGGGC-3'. Recent studies indicate that they also bind to the CACCC/GTGGG box (CAC/GT box), but less efficiently than to the GC box (32). Both GC and CAC/GT boxes are found in the regulatory regions of a variety of genes that are involved in cell growth and differentiation. Sp1 and Sp3 are ubiquitously expressed, whereas Sp4 is expressed mainly in brain and epithelial tissues (32). Sp1 regulates gene expression involved in cell proliferation and differentiation in various cell types (32). Sp1 acts as a basal transcription factor and facilitates the recruitment of TATA-binding protein and other transcriptional initiation complex machinery. The present study clearly indicates that, in addition to ESE-1, Sp1 positively regulates SPRR1B transcription. The –48/–39 bp promoter contains a DNA sequence (5'-GAGGCAGGGC-3') that is highly homologous to the Sp1 consensus sequence. The requirement of both proximal and enhancer elements, which cannot function independently, indicates the strong interdependence and cooperative interaction between ESE-1, Sp1, and AP-1 proteins. In support of this notion, other studies have suggested a potential role for a combinatorial interaction between AP-1, Sp1, and ESE-1 proteins in the regulation of early and late differentiation marker expression. For example, SPRR1A (15) and SPRR2A (16) transcription in keratinocyte differentiation is regulated by interaction between AP-1, ESE-1, and Sp1. Interestingly, overexpression studies indicated a dual role for Sp1 in regulating SPRR1B expression (Fig. 7). In the unstimulated state, it cooperatively interacts with ESE-1 and the TRE-bound AP-1 complex and enhances basal expression of the promoter. In contrast to the unstimulated state, Sp1 exerts a negative influence on PMA-inducible transcription. This interaction is likely contingent on two changes: (i) replacement of a Jun·Fra-2 complex with a Jun·Fra-1 complex and (ii) an increase in the c-Jun and Fra-1 levels and post-translational modification of Jun·Fra-1. The AP-1 complex composed of the Jun·Fra-1 complex preferentially interacts with ESE-1 to drive PMA-inducible transcription, in which Sp1 has a minimal role. When Sp1 is overexpressed, it reduces high affinity interactions between the Jun·Fra-1 and ESE complexes while promoting ESE-Sp1 interactions. This will probably lead to suppression of PMA-inducible transcription. Thus, Sp1 acts as a "yin-yang" regulator in the context of the SPRR1B promoter. Indeed, this type of differential regulation of gene transcription by Sp1 has been demonstrated (33).

Previously, we have shown that PMA enhances the in vivo protein-DNA interactions in BE cells at the distal TREs located at positions –139 and –109 compared with unstimulated cells (6). Moreover, this region acts as a classical enhancer because it strongly up-regulates the heterologous promoter activity in transient transfection assays (6). Furthermore, we have shown that PMA stimulation alters the protein binding to this region, which is mainly mediated by AP-1 dimers such as Jun·Fra-1 (7). Unlike enhancer TREs, PMA treatment did not significantly alter the footprinting at the proximal elements in both primary cultures and S6 cells (Fig. 1). Based on the in vivo footprinting data and the facts that deletion of distal TREs strongly suppresses both ESE-1- and Sp1-inducible SPRR1B transcription (Fig. 5) and that cotransfection of ESE-1 and c-Jun (but not ESE-1 and Sp1 or Sp1 and c-Jun) synergistically transactivates transcription (Figs. 6 and 7), we propose the following model for the transcriptional activation of SPRR1B in BE cells. As illustrated in Fig. 10A, in unstimulated BE cells, transcription factors that bind to the functional enhancer TREs are different. For example, we have recently shown that the enhancer is occupied by Jun·Fra-2 dimers in both unstimulated S6 and non-expressing A549 cells (7). Upon PMA stimulation, the enhancer is mainly occupied by Jun·Fra-1 dimers. Moreover, we have shown that overexpression of Fra-1 transactivates the SPRR1B promoter in S6 and H441 cells as well as in A549 cells (7, 13). In contrast, Fra-2 suppresses basal, PMA-, and c-Jun-enhanced SPRR1B transcription (7, 13). Interestingly, nuclear extracts isolated from SPRR1B-expressing and -non-expressing cells stimulated with PMA display strong binding of AP-1 family members to the consensus TRE (7, 13). Thus, preferential binding of Jun·Fra-1 dimers to the distal enhancer by a yet unknown mechanism probably facilitates the interaction between the enhancer and proximal promoter occupied by ESE-1 and Sp1, leading to enhanced transcription in the bronchial epithelium.



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FIG. 10.
Model for the transcriptional activation of SPRR1B by PMA in airway epithelial cells. Enh, enhancer; Pro, promoter; T, TREs; E, EBS; GC, GC box; ta, TATA box; white ovals, Jun proteins; black ovals, Fra-1; striped ovals, Fra-2; black triangles, ESE-1; white triangles, Sp1; diamonds, other transcription factors (TFs).

 

Alternatively, as shown in Fig. 10B, the increase in protein binding to the distal enhancer, as demonstrated by in vivo footprinting data, somehow facilitates interaction of AP-1 proteins with the other transcription factors such as ESE-1 and Sp1, which bind to the proximal promoter. ESE family members have unique A/T hook domains similar to those found in the high mobility group of proteins that bind to AT-rich regions (21). Based on the nature of the DNA bending properties of ETS family proteins (34), we speculate that the induction of SPRR1B expression is probably mediated by DNA looping between the enhancer and proximal promoter, resulting in assembly and/or entry of transcription factors to form the initiation complex, ultimately driving transcription (35). Consistent with this, a direct interaction of ETS proteins with the N-terminal domain of c-Jun has been demonstrated (36). Overexpression (Fig. 7) and EMSA (Fig. 8) also indicate that the relative ratio and distribution of Sp1 and ESE-1 proteins, as well as their mutually exclusive and/or inclusive interactions at the proximal promoter, play a critical role in the regulation of SPRR1B expression in human BE cells.

Although the exact mechanisms leading to the induction of squamous differentiation in the bronchial epithelium are unclear, the highly inducible nature of SPRR1B in BE cells by PMA, tobacco smoke, and other carcinogens provides a good model system to further elucidate the mechanisms of interaction between ESE-1, Sp1, and AP-1 proteins as well as other factors such as activators and coactivators governing such processes (37, 38). Moreover, a comparative transcription analysis of SPRR induction in bronchial epithelial cells and keratinocytes may provide additional insight into the mechanisms by which the squamous differentiation process is regulated in these two distinct cell types, which express mucous and squamous properties, respectively, under physiological conditions.


    FOOTNOTES
 
* This work was supported by National Institutes of Health Grants HL58122, HL66109, and ES011863. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ To whom correspondence should be addressed: Dept. of Environmental Health Sciences, Div. of Physiology, The Johns Hopkins University, Rm. W7006, 615 North Wolfe St., Baltimore, MD 21205. Tel.: 410-614-5442; Fax: 410-955-0299; E-mail: sreddy{at}jhsph.edu.

1 The abbreviations used are: BE, bronchial epithelial; PMA, phorbol 12-myristate 13-acetate; TRE, 12-O-tetradecanoylphorbol-13-acetateresponsive element; EBS, ETS-binding site; RT, reverse transcription; DMS, dimethyl sulfate; EMSA, electrophoretic mobility shift assay; PTBE, primary cultured tracheobronchial epithelial. Back


    ACKNOWLEDGMENTS
 
We thank the scientists referred to under "Materials and Methods" for providing the various expression vectors used in this study. We thank Dhan Kalvakolanu, Antonio Tugores, and Maureen Horton for helpful comments on this manuscript. We acknowledge the Urban Environmental Center of the Johns Hopkins University for use of its core facilities.



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