Perinatal regulation of the ClC-2 chloride channel in lung is mediated by Sp1 and Sp3

Shijian Chu, Carol J. Blaisdell, Min-Zhi M. Liu, and Pamela L. Zeitlin

Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287-2533


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Mechanisms responsible for regulation of pulmonary epithelial chloride-channel expression in the perinatal period are under investigation to better understand normal lung development and airway disease pathogenesis. The ClC-2 epithelial chloride channel is regulated by changes in pH and volume and is most abundant in lung during fetal development. In this study, we identify and sequence the ClC-2 promoter, which is GC rich and lacks a TATA box. By construction of a series of promoter-luciferase constructs, a 67-bp GC box-containing sequence in the promoter is shown to be critical to ClC-2 expression in primary and immortalized fetal lung epithelial cells. Electrophoretic mobility shift assays and antibody supershifts demonstrate that the Sp1 and Sp3 transcription factors are expressed in fetal lung nuclei and interact with the GC box sequences in the promoter. Immunoblotting techniques demonstrate that Sp1 and Sp3 are perinatally downregulated in the lung with the same temporal sequence as ClC-2 downregulation. This work suggests that Sp1 and Sp3 activate ClC-2 gene transcription and that reduction in Sp1 and Sp3 at birth explains perinatal downregulation of ClC-2 in the lung.

transcription; development; gene expression; ribonucleic acid polymerase II; epithelial cell


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ORCHESTRATED GENE EXPRESSION is critical to lung development. Many highly regulated genes are expressed in the lung during specific developmental stages. ClC-2 is one of several chloride channels that are present in the lung (32) and may participate in fluid regulation and homeostasis (17). Furthermore, this redundancy in ion channels makes it possible to manipulate an alternative chloride channel for therapeutic purposes (7). ClC-2 may be able to complement chloride transport diseases such as cystic fibrosis (CF). Therefore, studies of gene regulation will help us understand lung development and design new therapies for genetic diseases.

Our laboratory (23) recently demonstrated that rat lung ClC-2 expression is rapidly downregulated at birth. This downregulation occurs when the lung switches from chloride and fluid secretion to net fluid absorption, an important step for preparing the fetal lung for air breathing. It has been shown that fetal lung fluid secretion necessary for normal lung morphogenesis to occur is dependent on chloride secretion into the developing airways (31). The CF transmembrane conductance regulator (CFTR), the mutants of which cause postnatal airway disease, is a cAMP-regulated chloride channel expressed in the lung (21, 26). Because ClC-2 and CFTR are both found in the apical membrane of airway epithelial cells (22) and Schwiebert et al. (28) showed that overexpression of ClC-2 in CF bronchial epithelial cells in vitro complements the endogenous mutant CFTR, upregulation of ClC-2 expression in the postnatal lung could be used to functionally compensate mutant CFTR in CF patients.

Our initial screen of the sequence 5' to the ClC-2 gene uncovered several Sp1 binding sites in close proximity. The CFTR promoter also contains Sp1 binding sites (4). Sp1 is a zinc finger transcription factor that regulates many mammalian promoters (3, 10). For example, the Clara cell secretory protein (CCSP) gene, the surfactant protein B gene, and the uteroglobin gene, all expressed in the lung, are regulated by Sp transcription factors (9, 19, 33).

In this study, we characterize the ClC-2 promoter, define key elements in the promoter, and demonstrate regulation by the transcription factors Sp1 and Sp3. Furthermore, we show developmental regulation of Sp1 and Sp3 in the lung, which may explain the similar expression pattern of ClC-2 as well as the regulation of other developmentally dependent genes.


    MATERIALS AND METHODS
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MATERIALS AND METHODS
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Cell culture and plasmids. A pre-type II cell line (preII-19) derived from 19-day-gestation fetal rats was kindly provided by Dr. G. W. Hunninghake (University of Iowa, Iowa City, IA) (18). It was maintained in monolayer culture in DMEM with 4.5 g/l of glucose (Mediatech, Herndon, VA) supplemented with 10% fetal calf serum, 2.5 µg/ml of Fungizone, 100 U/ml of penicillin G, and 100 U/ml of streptomycin. The cells were fed three times a week and split 1:10 when they reached 100% confluence. The L2 cell line was derived from type II-like alveolar pneumocytes from normal adult female rat lung (American Type Culture Collection, Manassas, VA). It was maintained in monolayer culture in Ham's F-12K medium (Biofluids, Rockville, MD) containing 10% fetal calf serum, 2.5 µg/ml of Fungizone, 100 U/ml of penicillin G, and 100 U/ml of streptomycin. The cells were fed three times a week and split 1:10 when they reached 100% confluence.

Primary fetal distal lung epithelial (FDLE) cells were prepared from Sprague-Dawley rats (Harlan, Indianapolis, IN). Eighteen-day-gestation timed-pregnant dams were euthanized with CO2, and the fetuses were recovered by hysterectomy. Lungs from a single litter were pooled and placed in ice-cold Hanks' balanced salt solution (HBSS). Primary cells were isolated with a protocol modified from O'Brodovich et al. (24). Tissue was rinsed three times with cold HBSS, trachea and bronchi were removed, and whole lung segments were minced to 1-mm3 pieces. Tissue was digested with 0.125% trypsin (GIBCO BRL, Gaithersburg, MD) and 20 µg/ml of DNase (Worthington Biochemical, Freehold, NJ) in HBSS at 37°C, filtered through a 70-µm nylon cell strainer (Becton Dickinson, Franklin Lakes, NJ), and centrifuged to collect dissociated cells. Cells were resuspended and incubated in 0.1% collagenase and 20 µg/ml of DNase in Ham's F-12 medium (Mediatech) for 20 min at 37°C, then pelleted. Isolated cells were resuspended in Ham's F-12 medium containing 10% fetal calf serum, 100 U/ml of penicillin, 100 U/ml of streptomycin, and 2.5 µg/ml of Fungizone and plated on uncoated flasks for 0.5 h and again for 1 h to remove fibroblasts and enrich for epithelial cells. Cell viability was assessed by trypan blue exclusion with an inverted Olympus microscope and was >90%. Primary epithelial cells were seeded at 0.2-0.3 × 106 cells/cm2 and grown at 37°C in defined fetal epithelial cell medium, as previously described (34), consisting of Ham's F-12 medium, 7.5 µg/ml of endothelial cell growth supplement, 5 µg/ml of insulin, 7.5 ng/ml of transferrin, 0.1 µM hydrocortisone, 15 µg/ml of bovine pituitary extract (Collaborative Biomedical, Bedford, MA), and 20 ng/ml of cholera toxin (List Biological Laboratories, Campbell, CA).

Rat genomic clone 6369 (5) contains the entire upstream sequence presented in this study. DNA sequencing and template preparations were performed as previously described (5). DNA stem-loop structures and inverted repeats were analyzed by DNA Strider (version 1.2; Christian Marck).

Primer extension. Total RNA was prepared from 18-day-gestation fetal rat whole lung with the TRIzol Reagent (GIBCO BRL). Oligonucleotide 6369-9 was used as the primer. To label the primer, 10 pmol of the oligonucleotide were added in a 10-µl reaction containing T4 polynucleotide kinase buffer, 30 µCi of [gamma -32P]ATP, and 10 U of T4 polynucleotide kinase. The reaction was incubated at 37°C for 10 min and stopped by heating at 90°C for 2 min. The final concentration of the primer was adjusted to 100 fmol/µl by the addition of distilled water.

Primer-extension experiments were performed with a kit as described by the manufacturer (Promega, Madison, WI). In each reaction, the fetal lung total RNA was mixed with 100 fmol of radiolabeled oligonucleotide 6369-9 and the reaction buffer provided in the kit. The reaction was heated at 90°C for 2 min and slowly cooled down to room temperature over 1 h. Then 1.4 µl of 40 mM sodium pyrophosphate and 1 U of avian myeloblastosis virus RT were added, and the reaction volume was expanded to 20 µl with nuclease-free water and proportional reaction buffer. The tube was incubated at 41°C for 30 min. The reaction was stopped by adding 20 µl of loading dye and heating at 90°C for 10 min. The primer-extension products were resolved in an 8% polyacrylamide gel containing 7 M urea. The gel was dried and exposed to film (Hyperfilm MP, Amersham, Arlington Heights, IL).

RNase protection assay. Total RNAs were prepared from preII-19 and L2 cells with the Direct Protect kit (Ambion, Austin, TX) and TRIzol Reagent (GIBCO BRL). To prepare the antisense riboprobe, a 320-bp ClC-2 promoter fragment ending at the Not I site at the beginning of the coding sequence (Fig. 1) was cloned into pBluescript plasmid. The plasmid was then linearized by EcoR I at the 5'-end of the 320-bp insert and used as the template for in vitro transcription with an Ambion Maxiscript kit with T3 RNA polymerase and [alpha -32P]UTP. The riboprobe was purified by polyacrylamide gel electrophoresis.


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Fig. 1.   A: genomic nucleotide sequence of intervening region between ClC-2 and rat rRPB17 in opposite directions (GenBank accession nos. AF105003 and AF105004, respectively). Predicted amino acid sequences are shown beneath coding exons in both genes. Nos. at right, bp no. Positive numbers are used in both genes starting from 1st residue in coding sequences. Numbering in intervening sequence is relative to ClC-2 coding sequence; * at bottom, first residue in published ClC-2 cDNA;  ^, transcriptional start sites measured by primer extension. Lines and dots at top, stems and loops, respectively. Underlines, GC boxes; italics and underlines, CAT sequences; double underlines, oligonucleotide primers used for RT-PCR and primer extension; lowercase letters, Not I site at beginning of ClC-2 coding sequence used for generating riboprobe for RNase protection assay. B: alignment of rat RPB17 cDNA (top), human RPB17 cDNA (middle), and amino acid (bottom) sequences. Only different nucleotide residues are shown in human cDNA sequence. Dashed lines, identical bases. Amino acid sequence is identical between rat and human.

RNase protection assays were performed with the Direct Protect kit following the manufacturer's protocol. The products were resolved on a 5% polyacrylamide gel containing 8 M urea, and after the gel dried, it was exposed to film (Hyperfilm MP, Amersham) for 3-10 days.

Promoter-luciferase plasmid construction. The pGL3-basic vector (Promega) containing a promoterless luciferase gene was used to construct promoter clones. Some of the restriction sites used in subcloning are shown in Fig. 2. An 8-kb Not I fragment of the genomic clone 6369 (5) was initially cloned into Not I-digested pBluescript SK (pBN8). The insert was cut out with Sac I and Sal I and subcloned into Sac I- and Xho I-digested pGL3 (the 8-kb construct). Self-ligation of a Kpn I-digested 8-kb construct eliminated the most 5'-end part of the insert and yielded the 1.6-kb construct. The 990-bp construct was produced by cloning of a Spe I-Hind III fragment from the 1.6-kb construct into a Nhe I-Hind III-digested pGL3 vector. This construct was digested with Pst I and Nhe I, filled in to produce blunt ends, and self-ligated to yield the 990-67 bp construct. For the 428-bp construct, pBN8 was digested with Sac II and self-ligated. A Sac I-Sal I fragment was then cut out and subcloned into Sac I-Xho I-digested pGL3. A Pml I-EcoR V fragment from pBN8 was subcloned into Sma I-digested pGL3 to produce the 406-bp construct. An Nhe I-Bgl II fragment from the 428-bp construct was subcloned into Nhe I-Bgl II-digested pGL3, yielding the 170-bp construct. To obtain the 108-bp construct, a pBN8 clone with the insert in the reversed orientation was digested with Sma I and self-ligated. This clone contained only the 3'-end 108-bp ClC-2 sequence. The insert was cut out with Kpn I and Sac I and subcloned into Kpn I-Sac I-digested pGL3.


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Fig. 2.   ClC-2 promoter-luciferase gene constructs. A: restriction sites used for all promoter constructs except for original clone of 8 kb. Nos. on bottom, distance in bp upstream from first Met codon. B: nos. on right are lengths of corresponding ClC-2 promoter fragments in bp. Selected consensus protein binding sites are labeled.

Luciferase assays. ClC-2 promoter-luciferase gene constructs were introduced by lipofection to cell cultures at 70-80% confluence. Each ClC-2-luciferase plasmid was cotransfected with the beta -galactosidase expression plasmid (pCMV-SPORT-beta -gal; GIBCO BRL) for calibration of transfection efficiency. Two micrograms of each plasmid DNA and twenty microliters of lipofectamine (GIBCO BRL) were mixed in 0.5 ml of serum-free medium and incubated at room temperature for 30 min. Each 35-mm dish was washed with 1 ml of serum-free medium before a mixture of 0.5 ml of serum-free medium and 0.5 ml of DNA-lipofectamine was added, and the cells were returned to the CO2 incubator for 3 h. The solution was replaced with 1.5 ml of serum-containing medium, and the dishes were incubated for 2 days before being harvested.

Cells were washed with PBS once and incubated with 250 µl of the reporter lysis buffer (Promega) for 15 min at room temperature. The cells were then scraped and collected in microcentrifuge tubes on ice. After being vortexed for 15 s, the cell suspensions were centrifuged at top speed (16,000 g) in a microcentrifuge for 2 min at 4°C. The supernatants were collected and stored at -80°C until beta -galactosidase and luciferase activities were measured.

The beta -galactosidase enzyme assay system (Promega) was used for beta -galactosidase measurement according to the manufacturer's protocol. A luciferase assay system (Promega) was used for luciferase measurement. Readings were recorded on a Monolight 2010 luminometer (Analytical Luminescence Laboratory, Ann Arbor, MI). For each dish, duplicate measurements were taken for 20 s each. The mean of duplicates was then divided by the beta -galactosidase activity of the same dish, yielding the relative value of luciferase activity.

Preparation of nuclear extracts. Nuclear extracts were prepared as previously described (16) with some modifications. Approximately 1 × 108 preII-19 cells grown for 24 h were used for each preparation. The entire procedure was carried out at 4°C. Cells were collected and washed once with cold PBS and once with buffer A (10 mM HEPES-KOH, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, and 0.5 mM dithiothreitol). After centrifugation in a bench-top centrifuge at 3,000 rpm for 5 min, the cell pellet was resuspended in 0.5 ml of buffer A and kept on ice for 15 min. The cells were then lysed by four rapid ejections through a syringe attached with a 25-gauge, <FR><NU>5</NU><DE>8</DE></FR>-inch hypodermic needle. The crude nuclei were collected by a 20-s centrifugation at full speed in a bench-top centrifuge. The pellet was resuspended with 0.3 ml of buffer C (20 mM HEPES-KOH, pH 7.9, 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, and 0.5 mM dithiothreitol) and kept on ice for 30 min with occasional mixing. The suspension was then centrifuged at top speed in a bench-top microcentrifuge for 15 min. The supernatant was dialyzed for 4 h against two changes of buffer D (20 mM HEPES-KOH, pH 7.9, 20% glycerol, 0.1 M KCl, 0.2 mM EDTA, and 0.5 mM dithiothreitol). Insoluble material was removed by centrifugation, and aliquots were kept at -80°C.

Electrophoretic mobility shift assay. Complementary pairs of the indicated oligonucleotides (Table 1) were annealed in a 30-µl reaction volume containing 1 nmol of each oligonucleotide, 0.1 M Tris · HCl (pH 7.5), 0.5 M NaCl, and 0.05 M EDTA. The reaction was incubated at 65°C for 10 min and slowly cooled down to room temperature over a period of 1-2 h. The double-stranded oligonucleotide was then radiolabeled in a 10-µl reaction volume containing 3.3 pmol of oligonucleotide, 1 µl of 10× T4 polynucleotide kinase buffer (0.7 M Tris · HCl, pH 7.6, 0.1 M MgCl2, and 50 mM dithiothreitol), 1 µl of [gamma -32P]ATP (10 µCi), and 1 µl of T4 polynucleotide kinase (5-10 U). After incubation at 37°C for 10 min, 1 µl of 0.5 M EDTA and 89 µl of 10 mM Tris-1 mM EDTA buffer were added. Unincorporated nucleotides were eliminated from the labeled oligonucleotide by running the samples through a Sephadex G-25 minicolumn (Boehringer Mannheim, Indianapolis, IN). The incorporated radioactivity was measured on a scintillation counter. Sp1 oligonucleotide was purchased from Promega.

                              
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Table 1.   Oligonucleotides used in EMSA, primer extension, and PCR

In each binding reaction, 2 µl of nuclear extract were mixed with a radiolabeled oligonucleotide (104-105 counts/min) in a 10-µl reaction volume containing 10 mM Tris · HCl, pH 7.5, 1 mM MgCl2, 0.5 mM dithiothreitol, 4% glycerol, 0.5 mM EDTA, 50 mM NaCl, and 0.5 µg of poly(dI-dC) · poly(dI-dC). The reaction was incubated at room temperature for 20 min. When cold competitor was used, unlabeled oligonucleotide was added in 100× molar excess and incubated at room temperature for 10 min before addition of the labeled oligonucleotide. In gel supershift reactions, 1 µl of anti-Sp1 (or anti-Sp3) antibody (1 mg/ml; Santa Cruz Biotechnology, Santa Cruz, CA) was added after the 20-min incubation with the labeled probe, and the reaction was kept at room temperature for an additional 30 min. The same concentration of BSA was used as a control. The samples were loaded on a 4% nondenaturing polyacrylamide gel after they were mixed with a 10× loading buffer (250 mM Tris · HCl, pH 7.5, 0.2% bromphenol blue, and 40% glycerol). After electrophoresis, the gel was dried and exposed to film (Hyperfilm-MP, Amersham) from 1 to 3 days with intensifying screens.


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MATERIALS AND METHODS
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The ClC-2 promoter is GC rich and lacks a TATA box. The ClC-2 promoter limits were first defined by the identification of an open reading frame in the opposite direction 1,930 bp 5' to the first Met codon of ClC-2 (Fig. 1). The open reading frame corresponds to the human (h) RNA polymerase II subunit hRPB17 (see Identification of an RNA polymerase II subunit gene and transcript). Within this 1,930-bp region are three CAAT boxes, one of which is close to the ClC-2 coding sequence (-738 bp), and the other two at the middle (-1,101 and -1,176 bp; Fig. 1). Multiple GC boxes (10) were identified in this region. Four of them are within a 391-bp region upstream from the first Met codon of ClC-2. The first three GC boxes are highly conserved in human ClC-2 as reported by Cid et al. (6), with similar spacing between each other and in the same orientation. In addition, two potential stem-loop structures are present in this region. The first stem-loop structure overlaps with the first two GC boxes and the second stem-loop structure resides between the third and fourth GC boxes (Fig. 1). These structures may indicate a regulatory region for transcription initiation (8, 25). This area is also GC rich (75%). Consistent with the observation that promoters with GC-rich regions are usually TATA-less promoters, no TATA box was found in this 1,930-bp intervening region. In an area close to the RPB17 gene, three GC boxes were found within 500 bp of the first Met codon (Fig. 1). Similar to the sequence upstream of ClC-2, this region is also GC rich, containing 66% GC residues.

Primer-extension experiments were performed with oligonucleotide 6369-9 and total RNA prepared from fetal rat lung. The specificity of oligonucleotide 6369-9 was confirmed by sequencing and PCR. Sequencing of the genomic clone 6369 (~100 kb in size) (5) with oligonucleotide 6369-9 as the primer generated a high-quality sequence. PCR amplification of genomic DNA with primer pairs including oligonucleotide 6369-9 also generated fragments of expected sizes (data not shown). Two transcriptional initiation sites were observed in the primer extension (Fig. 3A). One was found 48 bp upstream from the first Met codon (103-bp product in Fig. 3) and the other was 39 bp 5' to the Met codon (the 94-bp product in Fig. 3) (Fig. 1). To confirm these sites, RNase protection assays were performed with RNA prepared from preII-19 and L2 cells (Fig. 3B). Two bands 50 and 59 bp in size matched the two initiation sites determined by primer extension. The top band in Fig. 3B, however, could also be a transcription initiation site, but it was not confirmed by primer extension. There could be other minor transcription initiation sites present because TATA-less promoters are often associated with multiple transcription initiation sites. In the experiments we performed, only two sites were confirmed. Although the untranslated RNA sequences at the 5'-ends are shorter than those found in human ClC-2 (6), similarities exist in that the downstream initiation site in human is also more predominant.


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Fig. 3.   A: primer extension in ClC-2 upstream region. Total RNA was prepared from fetal rat lung. Radiolabeled oligonucleotide 6369-9 was used as primer. Experimental procedure is described in MATERIALS AND METHODS. Nos. at left, size markers in bp. Arrows, sizes of primer-extension products representing lengths from 5'-end of transcripts to 3'-end of primer 6369-9. B: RNase protection assay. Probe was radiolabeled reverse-transcribed RNA derived from ClC-2 promoter sequence as described in MATERIALS AND METHODS. Lane 1: control without RNase cocktail; lane 2: control with no cellular RNA. Nos. at left, size markers in bp. Cellular RNA from pre-type II cells derived from 19-day-gestation fetal rats (preII-19) was added to reaction in lanes 1, 3, and 4, whereas L2 cellular RNA was added to reaction in lane 5. A standard amount of RNase cocktail (10 µl) was used in reactions in lanes 2 and 3, whereas one-fifth of RNase cocktail was used in lanes 4 and 5. Arrows, protected RNA fragments.

Identification of an RNA polymerase II subunit gene and transcript. By comparison with the hRPB17 cDNA sequence (29) (European Molecular Biology Organization accession no. Z49199) and the rat (r) RPB17 gene sequence, introns were identified between putative coding sequences. Three exons were revealed by sequence comparison. The deduced amino acid sequences from rat and human were 100% identical, and 91% identity was found when the rat and human cDNA nucleotide sequences were compared. Most of the variable residues were at the third position of codons (Fig. 1). RT-PCR was then performed on rat tissue cDNA to confirm the predicted exons and the expression of rRPB17. The 5' primer used in the PCR reaction was oligonucleotide 6369-20, located upstream from the predicted rRPB17 coding sequence. The 3' primer was oligonucleotide 6369-27-2, derived from the putative exon 3 of the predicted gene. Both oligonucleotides are indicated by a double underline in Fig. 1. A single fragment of ~400 bp was amplified from adult rat brain, kidney, and stomach cDNA preparations. Subsequent DNA sequencing of the amplicons demonstrated a cDNA sequence composed of the expected exons (Fig. 1). It also suggested that the 5'-end of the message is at or beyond the oligonucleotide 6369-20 priming site, which is 167 bp upstream from the predicted translational start.

A 67-bp sequence 5' to the gene is essential for highlevel ClC-2 transcription initiation. For functional studies of the ClC-2 promoter, a series of ClC-2 upstream sequences were obtained by deletions from the 5'-end of the 8-kb promoter clone with restriction enzymes (Fig. 2) and subcloned into the pGL3-basic vector (Promega) in front of a luciferase gene. The insert lengths in these clones vary from 108 bp to 8 kb as illustrated in Fig. 2. The luciferase activity was used as an indication of the strength of the transcriptional initiation of the inserted sequences.

Luciferase measurements demonstrated that a short sequence of 67 bp from 171 to 237 bp in the promoter region is necessary for high-level transcription initiation in a fetal lung epithelial cell line (preII-19) (Fig. 4A). The luciferase activity level was nearly baseline without this 67-bp sequence (in the clone of the 170-bp fragment), whereas inclusion of this sequence (the 237-bp construct) produced the highest luciferase measurement. Three GC boxes (B1-B3) were present within the 67-bp sequence. Inclusion of a more upstream sequence up to 428 bp decreased the promoter activity, suggesting that the sequence 5' to the 67 bp contained some negative regulatory elements. However, the 990-bp construct that contained another GC box (B5) again produced moderate promoter activity. Deletion of the 67-bp sequence from the 990-bp promoter significantly reduced the luciferase activity, again suggesting the importance of the 67-bp sequence in the promoter. The low-level luciferase activity from the 1.6-kb sequence may be due to the RPB17 gene promoter because it included one of the three GC boxes near the RPB17 gene. This sequence also contained two of the three CAT sequences in the middle of the intervening region. This may suggest that the two promoters affect each other.


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Fig. 4.   Relative luciferase activity produced by ClC-2 promoter-luciferase gene constructs in preII-19 cells (A) and primary fetal rat lung epithelial cells (B). beta -gal, beta -Galactosidase; nos. at bottom, lengths of corresponding ClC-2 promoter fragments in bp. Values are averages from 6 experiments in preII-19 cells and 8 experiments in primary cells. The most significant difference was found between 170-and 237-bp constructs, indicating importance of 67-bp sequence. The 237-bp promoter construct was significantly more active than all other constructs in both preII-19 and primary cells (P < 0.01 by 1-way ANOVA and Bonferroni multiple comparisons test).

The same transient transfection assay was also performed in primary rat FDLE cells. As shown in Fig. 4B, relative luciferase activity from the 237-bp promoter construct was much higher than other promoter constructs. The difference was more profound than that in the preII-19 cells. This suggests that in primary cells, the 237-bp fragment receives maximum activation and minimum inhibition. It is also likely that sequences 5' to the 237-bp fragment contain the targets for inhibitory factors that are more active or abundant in the primary cells.

Sp1 and Sp3 are involved in ClC-2 transcription initiation. Because multiple GC boxes were in the ClC-2 upstream region and the GC boxes may interact with regulatory proteins such as the Sp transcription factors (10, 11, 14), electrophoretic mobility shift assay (EMSA) was then performed to identify these protein factors.

Oligonucleotides derived from the ClC-2 promoter sequence were designed for EMSA to investigate interactions of the promoter with nuclear factors. The promoter region that interested us most was the 67-bp sequence that contains three GC boxes and produces the highest promoter activity in both preII-19 cells and primary lung epithelial cells. Oligonucleotide 54 resides at the 5'-end of the region and contains the third GC box, whereas oligonucleotide 53 is from the 3'-end of the region and contains the first and second GC boxes (Table 1, Fig. 5). Both oligonucleotides formed DNA-protein complexes after incubation with the preII-19 cell nuclear extract (Fig. 6A). Interestingly, the band patterns of these two oligonucleotides in EMSA were highly similar. Four distinct bands were seen in reactions with these oligonucleotides. Competition analysis that used a heterologous oligonucleotide containing a consensus GC box sequence (Sp1; Promega) diminished bands 1-3 (Fig. 6A), suggesting that the GC boxes might be important for the complex formation in these three bands. To confirm this observation, a mutant oligonucleotide of oligonucleotide 54 was synthesized in which two base pairs in the GC box were changed from GG to AT (oligonucleotide 541; Table 1). As expected, this mutation significantly decreased the binding of the oligonucleotide to the protein factors in bands 1-3 (Fig. 6A). The fourth band, which was apparent in reactions with both probes, was not affected by the unlabeled oligonucleotide Sp1 and the mutant GC box in oligonucleotide 54. It is therefore not related to the GC box sequence. The protein factor present in this band is still not known.


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Fig. 5.   Schematic drawing of locations of oligonucleotide probes used in electrophoretic mobility shift assays (EMSAs). Top line represents part of ClC-2 promoter sequence. Nos. on top, positions relative to ClC-2 translation start site; open boxes, GC boxes. Lines at bottom, oligonucleotide probes used in EMSA with names on left.


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Fig. 6.   A: EMSAs with nuclear extract from preII-19 cells. Top: radiolabeled oligonucleotide probes used in each reaction, unlabeled Sp1 probe used in competition assays, and antibodies against Sp1 and Sp3 used in supershift reactions. +, With; -, without. Nos. at left, bands before supershift (see text). B: EMSA under same conditions as in A but with no unlabeled Sp1 probe and antibodies.

Because a number of transcription factors of the Sp1 multigene family bind GC boxes, we tested the presence of Sp1 and Sp3 by antibody supershift. Again, similar patterns were seen with both oligonucleotide 53 and 54 probes. Band 1 was supershifted by anti-Sp1, and bands 2 and 3 were supershifted by anti-Sp3 (Fig. 6A). This was consistent with the observation in competition assays that bands 1-3 all contained GC box binding proteins. Apparently, Sp1 was present in the band 1 complex, whereas both complexes of bands 2 and 3 contained Sp3. The Sp3 in the fast migrating band might be a truncated form (1).

To separate the binding activity of the first and second GC boxes in oligonucleotide 53, new oligonucleotide probes B1 and B2 were prepared to examine these GC boxes individually (Fig. 5). Further EMSA demonstrated that the probe containing the first GC box had no binding activity to the protein factors in bands 1-3 as seen with oligonucleotide 53, whereas the probe containing the second GC box showed the same binding activity as oligonucleotide 53 (Fig. 6B). Therefore, the protein-DNA complexes formed with oligonucleotide 53 were essentially due to the upstream sequence in the oligonucleotide containing the second GC box.

Two additional GC boxes are present upstream from the 67-bp sequence. One of them is in the 990-bp fragment that produced the second highest promoter activity in preII-19 cells. Gel supershift experiments with oligonucleotides containing these GC boxes showed that both Sp1 and Sp3 could bind these sequences (Fig. 7).


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Fig. 7.   Binding of Sp1 and Sp3 to GC box-containing sequences in ClC-2 promoter region shown by gel supershift reaction. Nos. at left, bands before supershift reaction.

Sp1 and Sp3 are developmentally regulated in lung. Because ClC-2 lung expression is developmentally downregulated (23) and is dependent on Sp binding sites, we asked whether Sp factors are also developmentally downregulated. Immunoblotting was performed with rat lung homogenates from different developmental stages. Equal protein was loaded by protein concentration as determined with the Bio-Rad DC protein assay reagents (Hercules, CA), Coomassie blue staining of the gel (data not shown), and probing with anti-beta -actin antibody (clone AC-15, Sigma, St. Louis, MO; Fig. 8). Both Sp1 and Sp3 were significantly downregulated from 21 days of gestation and beyond (Fig. 8). The patterns of Sp1 and Sp3 expression in the lung are distinct but remarkably similar to ClC-2 and suggest that the Sp family of proteins, in association with other nuclear proteins, may be the critical factors in perinatal ClC-2 regulation.


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Fig. 8.   Immunoblots of rat lung homogenates from different developmental stages (17-, 19-, and 21-day fetal gestation, newborn, and adult). Each lane was loaded with 25 µg of homogenate. After transblotting, top part of membrane was reacted with 1:500 diluted rabbit anti-Sp1 (A) or rabbit anti-Sp3 (B) and bottom part was reacted with 1:500 diluted monoclonal anti-beta -actin (clone AC-15). Nos. at left, size in bp. According to manufacturer, Sp1 and Sp2 antibodies do not cross-react with each other or with other Sp factors. C: dephosphorylation of Sp1 by alkaline phosphatase (AP). In each reaction, 40 µg of 19-day-gestation rat lung homogenate were mixed with various amounts of AP in buffer provided by manufacturer. Arrows, phosphorylated and dephosphorylated Sp1. Reactions were incubated at room temperature for 2 h. Sp1 was detected by same anti-Sp1 antibody.

Sp1 activity is regulated by phosphorylation (12), and at least some phosphorylation decreases its DNA binding activity (2). We observed the typical doublet and converted the upper band to the lower band by in vitro dephosphorylation (Fig. 8C). The lower band of the Sp1 doublet was predominant in the 17-day fetal lung (Fig. 8A). The ratio of upper to lower band amounts constantly increased until it reached ~1 at birth. This indicates that Sp1 phosphorylation is also perinatally regulated.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Gene expression of many ClC chloride channels is highly regulated. Tissue-specific expression is observed for ClC-1 and ClC-3, which are uniquely expressed in skeletal muscles (30) and abundantly expressed in the brain (13), respectively. ClC-2 is more widely expressed, but expression is downregulated at birth in many organs (22).

The ClC-2 promoter belongs to a TATA-less class of promoters. Promoters of this class have been found in genes encoding proteins for cellular metabolism as well as growth factors and their receptors. Multiple transcriptional start sites and GC boxes are common features of this class of promoters (3). Another chloride-channel gene, CFTR, is very similar with respect to the promoter. Like the ClC-2 promoter, the CFTR promoter lacks a TATA box, has GC boxes (4) and multiple transcriptional start sites (4, 15), and drives low-level transcription. There may be common themes to regulation of the CFTR and ClC-2 genes.

Because ClC-2 expression in the lung is higher at fetal stages, we used the fetal lung epithelial cell line preII-19 in the luciferase reporter gene assays and EMSA. In the preII-19 cells, the highest transcriptional initiation was achieved by the 237-bp promoter, whereas the 170-bp sequence yielded almost background level transcription. We attribute this difference to the 67-bp sequence, which is absent in the 170-bp clone. The importance of this short sequence is supported by the fact that deletion of this 67-bp fragment from the 990-bp clone caused a significant decrease of its transcription efficiency. Three clustered GC boxes are the predominant feature of this short sequence. Similarly, in the CFTR gene promoter, the promoter fragments containing GC boxes conduct the highest transcription efficiency, but Sp1 and Sp3 binding have not been studied (4).

Whereas the GC box sequence GGGCGG has been regarded as the consensus binding sequence for the Sp transcription factors, other factors can affect Sp1 and Sp3 binding. In oligonucleotide B1, there is an uninterrupted string of GC residues around the consensus GC box (underlined in Table 1). However, EMSA showed that Sp1 and Sp3 had nearly no binding activity to this probe, which could be due to interference by other DNA binding proteins. Our results indicate that the GC boxes in the ClC-2 promoter, especially B2, B3, and B5, are important for transcription initiation because they are bound by Sp1 and Sp3 and are present in the two most active promoter constructs, i.e., 237 and 990 bp.

Because Sp1 and Sp3 are major transcription activators for ClC-2, we suspected that Sp1 and Sp3 may also be developmentally downregulated. Although the age dependence of Sp1 mRNA expression after birth has been reported (27), perinatal changes in Sp1 expression at the protein level have never been examined. Our finding of the developmental regulation of Sp1 expression and phosphorylation together with the regulation of Sp3 expression suggest a possible mechanism for ClC-2 downregulation. This is also consistent with the previous observation that Sp1 is important for early embryonic development (20). Because we used whole lung tissue homogenates in the immunoblotting, we do not yet know whether downregulation of ClC-2, Sp1, and Sp3 occurs in the same types of cells. However, we do know that Sp1, Sp3, and ClC-2 are expressed in the preII-19 cells. Phosphorylation of Sp1 affects its DNA binding and transactivating activity. If unphosphorylated Sp1 had higher activity, as observed in other promoters (2, 35), its proportional decrease during development would support its regulatory role in ClC-2 downregulation. The significance of higher-level fetal expression of Sp1 may well be more general in lung development and organogenesis because it regulates a number of other pulmonary genes (9, 19, 33), and knockout of Sp1 results in significant disruption of embryogenesis (20). Whether Sp1 has a similar perinatal expression pattern in other organs remains unanswered.

This study is the first report on transcriptional regulation of a chloride channel in lung epithelial cells. The maximal level of ClC-2 transcription mediated by Sp1 and Sp3 together with the striking downregulation of Sp1 and Sp3 expression at birth may explain the perinatal downregulation of ClC-2. Because Sp1 and Sp3 regulate many other genes, some of which are growth regulated, perinatal downregulation of Sp1 and Sp3 may play a critical role in organogenesis and maturation of the lung and possibly other organs as well.


    ACKNOWLEDGEMENTS

We thank Dr. G. W. Hunninghake (University of Iowa, Iowa City, IA) for the pre-type II cell line.


    FOOTNOTES

This work was supported by grants from the Cystic Fibrosis Foundation (to P. L. Zeitlin) and the National Heart, Lung, and Blood Institute (to P. L. Zeitlin and C. J. Blaisdell) and a fellowship from the Cystic Fibrosis Foundation (to S. Chu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: S. Chu, Park 316, Johns Hopkins Hospital, 600 N. Wolfe St., Baltimore, MD 21287-2533 (E-mail: shijian{at}welchlink.welch.jhu.edu).

Received 22 September 1998; accepted in final form 5 January 1999.


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Am J Physiol Lung Cell Mol Physiol 276(4):L614-L624
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