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Published ahead of print on April 24, 2003, doi:10.1165/rcmb.2003-0030OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 29, pp. 499-505, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2003-0030OC

Modulation of Sp1 and Sp3 in Lung Epithelial Cells Regulates ClC-2 Chloride Channel Expression

Kathryn W. Holmes, Russell Hales, Shijian Chu, Micah J. Maxwell, Peter J. Mogayzel, Jr. and Pamela L. Zeitlin

Eudowood Division of Pediatric Respiratory Sciences, The Johns Hopkins Medical Institutions, Baltimore, Maryland

Address correspondence to: Dr. Pamela L. Zeitlin, Eudowood Division of Pediatric Respiratory Sciences, The Johns Hopkins Medical Institutions, 600 N Wolfe St. Park 316, Baltimore, MD 21287–2533. E-mail: pzeitlin{at}jhmi.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ClC-2 is a pH- and voltage-activated chloride channel, which is highly expressed in fetal airways and downregulated at birth. The ClC-2 promoter contains consensus binding sites within the first 237 bp, which bind transcription factors Sp1 and Sp3(1). This study directly links Sp1 and Sp3 with ClC-2 protein expression by demonstrating: (i) induction of ClC-2 protein by transient overexpression of each transcription factor in adult rat Type II cells, which have low levels of ClC-2; and (ii) reduction of ClC-2 expression by incubation with a competitive inhibitor of Sp1 and Sp3 in fetal rat Type II cells, which have high levels of endogenous ClC-2. Endogenous fetal lung Sp1 is differentially expressed as two major species of 105 kD and 95 kD. Although low-level expression of Sp1 in adult cells is almost exclusively the 105-kD species, overexpression of Sp1 results in increased expression of the 95-kD band. These experiments suggest that the mechanism for postnatal reduction of ClC-2 expression in lung epithelia is based on decreased interaction of Sp1 and Sp3 with the ClC-2 promoter.

Abbreviations: bovine serum albumin, BSA • cystic fibrosis, CF • CF transmembrane conductance regulator, CFTR • Dulbecco's phosphate-buffered saline with calcium and magnesium, DPBS • sodium dodecyl sulfate–polyacrylamide gel electrophoresis, SDS-PAGE


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The ClC-2 chloride channel is one member of a large superfamily of chloride channels (2). It is pH-, voltage-, and osmotically regulated (3–5). ClC-2 has been localized to epithelial cells lining the respiratory tract, small intestine, and kidney (6–8), and has been shown to contribute to chloride secretion and volume homeostasis in these tissues (7, 9–11). Interestingly, these regions also express the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) chloride channel. Our laboratory has shown that, like CFTR, ClC-2 is highly expressed in fetal airways and is rapidly downregulated at birth (12). This downregulation also occurs simultaneously with upregulation of the epithelial sodium channel (ENaC) when the lung switches from fluid secretion to air exchange (13, 14). Given the similarities between ClC-2 and CFTR, ClC-2 is a potential therapeutic target in patients with mutations in the CFTR gene (9). Importantly, ClC-2 has already been shown to act as an alternative pathway for chloride conductance when overexpressed in human CF bronchial epithelial cells (15).

The ClC-2 promoter belongs to a TATA-less class of promoters and contains multiple GC boxes. We have shown these GC boxes avidly bind Sp1 and Sp3 transcription factors from rat lung nuclei (1). In addition, the developmental expression patterns for Sp1 and Sp3 parallel that of ClC-2 (1), suggesting that the bulk amount of transcription factor might control ClC-2 mRNA expression. We hypothesize that, in addition to the absolute level of transcription factor expression, the isoform of Sp1 is a factor in determining the level of ClC-2 mRNA expression.

This study was designed to selectively overexpress Sp1 and Sp3 in L2 cells (immortalized adult rat Type II epithelial cells) that normally express low endogenous levels of Sp1, Sp3, and ClC-2. Protein expression was measured by immunoblotting and visualized by immunofluorescence. The reverse experiment, inhibition of the interaction of transcription factor with promoter, was designed to see a reduction in ClC-2 protein expression. The phosphorylation status of endogenous and transfected Sp1 and Sp3 was investigated and then manipulated in vitro. We show that the quantity of Sp1 and Sp3 and the isoform of Sp1 are important in regulation of ClC-2 expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
The L2 cell line was originally derived from type II–like alveolar pneumocytes from normal adult female rat lung (#CCL-149; American Type Culture Collection, Manassas, VA). A fetal or pre-type II cell line (preII-19), derived from 19-d-gestation fetal rats, was kindly provided previously by Dr. G. W. Hunninghake (University of Iowa, Iowa City, IA). Both cell lines were maintained as previously described (1). The pCMV-Sp1 plasmid was a kind gift from Dr. Robert Tjian, (Howard Hughes Institute, Berkeley, CA). The pCMV-Sp3 plasmid was a a kind gift from Dr. Guntram Suske (University of Marburg, Germany). The pRL-CMV control plasmid was obtained from Promega (San Louis Obispo, CA). Peptides for making ClC-2 antisera were ordered from Alpha Diagnostics, Inc. (Denver, CO). Tissue culture reagents and transfections kits were purchased from Life Technologies (Grand Island, NY). The antibiotic mithramycin A was purchased from Sigma (St. Louis, MO). Antibodies to Sp1 (SC-59) and Sp3 (SC-450) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Secondary antibodies and chemiluminescence system for immunoblotting were obtained from Amersham Pharmacia Biotech (Piscataway, NJ). Nitrocellulose membranes, solutions required for protein quantification, and densitometry software were obtained from Bio-Rad (Hercules, CA). All statistics were performed using SPSS software (SPSS, Inc., Chicago, IL).

For immunohistochemistry, fluorescein isothiocyanate secondary antibody and 4',6'-diamidine-2'phenylindole dihydrochloride were from Jackson ImmunoReserch (Westgrove, PA). Slowfade antifade reagent from Molecular Probes (Eugene, OR) was used to decrease light sensitivity. Specimens were examined on a Zeiss Axiovert 135 fluorescence microscope equipped with a 40x Plan Neofluar oil immersion objective lens (Carl Zeiss, Inc, Thornwood, NY), Chroma HQ fluorescence filter sets (Chroma Filters, Inc., Brattleboro, VT), and a CoolSnap HQ cooled CCD camera (Roper Scientific, Inc., Tucson, AZ). Images were acquired and processed with IPLab software (Scanalytics, Inc., Fairfax, VA).

Antibody to rCLC-2
Two peptides were constructed based on sequence predicted from rat genomic ClC-2. The peptide S787 was made from a region flanking exon 20 with the following sequence: STTSESDSDLC. This region is thought to be an area of alternative splicing (16). The peptide L785 was from exon 20, and had the following sequence: LEKSESCDKRKLKRVRISLA. Peptides were conjugated to KLH to increase immunogenicity. Rabbits were injected with peptide S787 or L785, and antiserum was extracted at monthly intervals. These antisera detect a single band at 101 kD in immunoblots of whole cell lysates in L2 and preII-19. The S787 was subsequently used for the experiments described in this article.

Overexpression of Sp1 and Sp3 via Transfection
Cells were transfected with Lipofectamine Plus using the manufacturer's protocol. Briefly, 1 d before transfection, L2 cells were seeded onto 100-mm plates and incubated at 37°C and 5% CO2 overnight to allow them to reach 60–80% confluence. Cells were then washed twice with Hanks' Balanced Salt Solution, and incubated for 1 h with 5 ml of OPTI-MEM reduced serum medium. Four micrograms of pCMV-Sp1 plasmid or pCMV-Sp3 were used for each transfection. Controls were transfected with Lipofectamine Plus without DNA, or with control plasmid pRL-CMV. Total transfection volume was 6.5 ml for each plate. At 4 h after transfection, 2 ml of regular growth medium were added to the transfection mixture. At 24 h after transfection, the medium was replaced with 8 ml of regular growth medium containing antibiotics and serum. Cells were collected at 24, 48, 72, and 96 h, and protein was quantified as previously described (17).

Interruption of Transcription Factor Binding
PreII-19 cells were passaged to 50% confluence in T25 flasks. The following day, medium was changed to one containing mithramycin A at concentrations of 25 or 75 nM. Medium without mithramycin A was used as control. After 72 h in the presence of mithramycin A, cells were harvested and proteins were analyzed by immunoblotting.

Immunoblotting
Ten to fifteen micrograms of whole cell lysate were diluted with RIPA (50 mM Tris-HCl, pH 8, 150 mM NaCl, 1% Triton X-100, 1% sodium dodecyl sulfate [SDS]) with the following protease inhibitors: 0.1 mM phenylmethylsufonylfluoride, 10 µg/ml aprotenin, 1 mM Na orthovanadate, and 5 mM ethylenediaminetetraacetic-acid. Five microliters of standard loading buffer was added to each sample for a total volume of 25 µl. Samples were heated for 30 min at 37°C before being loaded onto a 10% SDS-polyacrylamide gel (SDS-PAGE). Separated proteins were transferred to nitrocellulose membrane using ice-cold Towbin buffer. Nonspecific binding was blocked by incubation of the nitrocellulose with 5% dry milk in TBS–0.05% Tween 20 (Sp1 or Sp3 antibodies), or 2% nonfat dry milk and 1% bovine serum albumin (BSA) in phosphate-buffered saline–0.05% Tween 20 (ClC-2 antibody). Membranes were incubated overnight at 4°C with primary antibody for Sp1, Sp3, or ClC-2 antibody (S787) at a 1:1,000 dilution for Sp1 and ClC-2 and 1:500 dilution for Sp3. At the end of the primary incubation, blots were washed for 15 min twice and 5 min twice in blocking solutions without BSA or milk. A secondary anti-rabbit antibody at a dilution of 1:1,000 was applied for 1 h. Blots were then washed again as above. Detection of immunoreactivity was performed with enhanced chemiluminescence reagent and fluorography.

Immunohistochemistry
One day before transfection, coverslips were placed onto 6-well plates and coated with 1% collagen, 1% BSA, and 1% fibronectin. L2 cells were seeded onto plates and transfected as described above. Two micrograms of plasmid DNA was used for transfections.

At 24, 48, 72, and 96 h, coverslips were rinsed with Dulbecco's phosphate-buffered saline with calcium and magnesium (DPBS) twice. They were then fixed in cold 100% acetone and incubated at 4°C for 9 min. Cells were rinsed three times in DPBS, permeabilized in 2% Triton X-100 for 10 min at room temperature, washed again, then blocked with DPBS/5% goat serum. Cells were incubated overnight with Sp1, Sp3, or ClC-2 antibodies at a 1:200 dilution at room temperature in a humidity chamber. The following day, cells were washed, and fluorescein isothiocyanate–labeled anti-rabbit secondary antibody was applied at a 1:500 dilution. Cells were counterstained with 4',6'-diamidine-2'phenylindole dihydrochloride. Cells were washed again and overlaid with glycerol-PBS Slowfade antifade reagent. Specimens were examined on a Zeiss fluorescence microscope at x40. Exposure times were first determined with transfected slides, then held constant for the remainder of the experiment. A 95% normalization and a sharpen hat filter were applied to each slide. Images were then formatted to 8 bit and transferred into Microsoft Power Point.

Statistics
Quantification of protein expression was made by densitometry analysis with Quantity One as previously described (17). The relevant bands for ClC-2, Sp1, and Sp3 were normalized to the untransfected control. For ClC-2 data, controls were obtained for each time point. ANOVA was performed to test the significance of Sp1, Sp3, and mithramycin A data. For ClC-2, a Mann-Whitney test was used for comparison of transfected versus untransfected cells at each individual time point for the pCMV-Sp1 and pCMV-Sp3 transfections.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
S787 Recognizes a Single Band of ClC-2 at 101 kD
Polyclonal antiserum was raised in rabbits against a synthetic peptide spanning a deletion in ClC-2 that results from alternate splicing and is referred to as the "short-form 2a" (18). It is 20 amino acids shorter than full-length ClC-2. Antisera to both long (L785) and short (S787) detect the same 101-kD band (data not shown). A shorter 99-kD form of ClC-2 was not detected in these cell culture experiments. Endogenous ClC-2 expression from whole cell lysates was more abundant in the fetal preII-19 cell line than the adult L2 line (Figure 1A). Preincubation of the S787 rabbit antisera with immunizing peptide resulted in elimination of this 101-kD band in L2 (Figure 1B). Preincubation of L785 also resulted in elimination of the 101-kD band (data not shown).



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  		Figure 1. Anti–ClC-2 antiserum recognizes a 101-kD band that is eliminated by competition with excess peptide. Whole cell lysates were separated on a 10% SDS-PAGE gel as described in MATERIALS AND METHODS and evaluated by immunoblotting. (A) Fifteen micrograms of protein from preII-19 and L2 cells expressed the endogenous form of ClC-2 as a 101-kD band. (B) Ten micrograms of preII-19 protein was incubated by S787 antibody (first lane) or S787 antibody preincubated with neutralizing peptide (second lane).

 
Transient Overexpression of Transcription Factor Sp1 Increases Expression of ClC-2
Our hypothesis is that transient overexpression of Sp1 will lead to enhanced interaction at the consensus binding site(s) within the ClC-2 promoter and result in synthesis of ClC-2 protein. Adult rat L2 cells transfected with pCMV-Sp1 demonstrate robust overexpression of the 95-kD isoform of Sp1 (Figure 2A). Sp1 expression levels were significant at 24 h after transfection, and declined by 96 h (P < 0.01, n = 5, Figure 2B). Concurrently, these same lysates were probed with anti–ClC-2 antibody (S787), which identified a 101-kD protein (Figure 2C) that we have identified as ClC-2. As hypothesized, L2 cells overexpressing Sp1 significantly increased expression of ClC-2 at 48 and 72 h (P < 0.05, n = 6, Figure 2D). Of note, the increase in ClC-2 expression slightly lagged behind Sp1 overexpression, suggesting a temporal relationship between expression of Sp1 and ClC-2.



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  		Figure 2. Induction of ClC-2 protein expression by transfection of pCMV-Sp1 in L2 cells. Fifteen micrograms of protein from L2 whole cell lysates was separated on a 10% SDS-PAGE gel and evaluated by immunoblotting as described in MATERIALS AND METHODS. (A) Sp1 was expressed as 105- and 95-kD bands in untransfected cells at 48 h and in pCMV-Sp1 transfected cells at 24, 48, 72, and 96 h after transfection. (B) Densitometric analysis of repeated experiments (n = 5, *P < 0.01). (C and D) Concurrent ClC-2 protein expression in the same lysates (*P < 0.05, n = 6).

 
Transient Overexpression of Transcription Factor Sp3 Increases Expression of ClC-2
To test the hypothesis that the transcription factor Sp3 can upregulate ClC-2, adult rat L2 cells were transiently transfected with pCMV-Sp3. Similar to the data for Sp1, Sp3 protein expression increased by 24 h, peaked at 48 and declined by 96 h (P < 0.01, n = 5; Figures 3A and 3B). Transfected L2 lysates were concurrently probed with the S787 antisera to ClC-2. Increased ClC-2 expression was observed at 24 h after transfection, and became significant at 48 and 72 h (P < 0.05, n = 7; Figures 3C and 3D). These results suggest that transient overexpression of Sp3 leads to increased ClC-2 expression. Although the pattern of expression between Sp3 and ClC-2 mimics that of the pCMV-Sp1 transfections, the increased ClC-2 expression is less robust with Sp3 than with Sp1. The origin of this difference could be decreased transfection efficiency of pCMV-Sp3, decreased binding affinity of Sp3, interaction with other regulatory sites within the ClC-2 promoter, or competition from other transcription factors. As seen with the pCMV-Sp1 transfections, the increase in ClC-2 expression lags behind overexpression of Sp3 by ~ 24 h. These data suggest that increased Sp3 expression leads to the subsequent increase in ClC-2 expression patterns.



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  		Figure 3. Induction of ClC-2 protein expression by transfection of pCMV-Sp3 in L2 cells. Fifteen micrograms of protein from L2 whole cell lysates was separated on a 10% SDS-PAGE gel and evaluated by immunoblotting as described in MATERIALS AND METHODS. (A) Sp3 expression is demonstrated as 105- and 95-kD bands in untransfected cells at 48 h and in pCMV-Sp3 transfected cells at 24, 48, 72, and 96 h after transfection. (B) Densitometric analysis of repeated experiments (n = 6, *P < 0.01). (C and D) Concurrent ClC-2 protein expression with the same lysates (*P < 0.05, n = 7).

 
Transfection of pRL-CMV Does Not Lead to an Increase in ClC-2
It is possible that any gene expressed from a CMV promoter might lead to increased expression of ClC-2. To control for this possibility, we transfected 100-mm plates with reagent only, pRL-CMV, pCMV-Sp1, and pCMV-Sp3. Cells were harvested at 72 h, the peak of ClC-2 expression. We found an increase in ClC-2 expression with transfection of pCMV-Sp1 and pCMV-Sp3, but not with pRL-CMV (Figure 4). Therefore, the increased expression pattern of ClC-2 with transfection of pCMV-Sp1 and pCMV-Sp3 but not pRL-CMV is more likely to be a response to overexpression of Sp1 and Sp3 than a nonspecific transfection effect.



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  		Figure 4. Induction of ClC-2 protein expression is specific to pCMV-Sp1 and pCMV-Sp3. L2 cells were transfected as described in MATERIALS AND METHODS with lipofectamine reagent alone, pCMV-SP1, pCMV-SP3, or pRL-CMV. After 72 h, 15 µg of protein from whole cell lysates was separated on a 10% SDS-PAGE gel and evaluated by immunoblotting with S787 as described in MATERIALS AND METHODS. ClC-2 protein expression was increased with pCMV-SP1 and pCMV-SP3, but not with lipofectamine reagent or with a control Ranella plasmid pRL-CMV.

 
Immunofluorescence Imaging of pCMV-Sp1 and pCMV-Sp3 Transfections Support the Hypothesis that Sp1 and Sp3 Regulate ClC-2 Expression
To confirm that overexpression of Sp1 and Sp3 proteins leads to an increase in ClC-2 expression, we directly visualized the effect of pCMV-Sp1 and pCMV-Sp3 transfections with immunofluorescence techniques. L2 cells were seeded onto plates containing multiple coverslips and transfected with either pCMV-Sp1 or pCMV-Sp3. After 48 h, cultures were fixed and probed with antisera to Sp1, Sp3, or ClC-2. Immunofluorescence imaging is shown in Figures 5 and 6. There was a marked increase in signal corresponding to Sp1 in L2 cells transfected with pCMV-Sp1 as compared with untransfected controls (Figures 5A and 5B). Transfection efficiency was ~ 70% for pCMV-Sp1. Under the same experimental conditions, a similar percentage of cells transfected with pCMV-Sp1 also demonstrated a concurrent increase in ClC-2 at 48 h as compared with untransfected controls (Figures 5C and 5D).



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  		Figure 5. Imaging of pCMV-Sp1–driven induction of ClC-2. L2 cells were grown to 50% confluence and then transfected with pCMV-Sp1 as described in MATERIALS AND METHODS. (A) Arrows point to transfected cells with increased nuclear expression of Sp1 (red) at 48 h after transfection. (B) Untransfected cells expressing endogenous Sp1 at the same time point. (C) Induction of ClC-2 (green) expression in pCMV-Sp1 transfected cells was markedly increased at 48 h after transfection, as compared with endogenous ClC-2 levels in D.

 


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  		Figure 6. Imaging of pCMV-Sp3–driven induction of ClC-2. L2 cells were grown to 50% confluence and then transfected with pCMV-Sp3 as described in MATERIALS AND METHODS. (A) Arrows point to transfected cells with increased nuclear expression of Sp3 (magenta) at 48 h after transfection. (B) Untransfected cells expressing endogenous Sp3 at the same time point. (C) Induction of ClC-2 (green) expression in pCMV-Sp3 transfected cells was markedly increased at 48 h after transfection as compared with endogenous ClC-2 levels in D.

 
Figure 6 displays images from a similar experiment using pCMV-Sp3. Transfected cells demonstrated a marked excess of nuclear staining for Sp3 as compared with untransfected controls (Figures 6A and 6B). Transfection efficiency for pCMV-Sp3 was ~ 50–60%. In addition, the ClC-2 expression in pCMV-Sp3 transfected cells was increased as compared with untransfected controls (Figures 6C and 6D). In the absence of primary antibody, neither transfected nor untransfected cells demonstrated any specific nuclear or cytoplasmic staining (data not shown). These data confirm our immunoblot data demonstrating that Sp1 or Sp3 overexpression leads to increased ClC-2 expression.

Mithramycin A Decreases Expression of ClC-2
Our data suggest that Sp1 and Sp3 can lead to increased expression of ClC-2. We tested the converse hypothesis, that interference in the interaction of Sp1 or Sp3 with the ClC-2 promoter would reduce ClC-2 expression. PreII-19 cell line was selected for mithramycin A treatment because it has a high level of endogenous ClC-2 expression. Mithramycin A has affinity for the GGGCGG binding sequence, and effectively blocks the binding of Sp1 and Sp3 (19–21). We anticipated that mithramycin A would block Sp1 and Sp3 binding and inhibit endogenous ClC-2 protein expression. PreII-19 cells were exposed for 72 h in media containing increasing concentrations of mithramycin A. Treatment of preII-19 cells with 75 nM mithramycin A significantly reduced ClC-2 expression by 50% (P < 0.001, n = 8, Figures 7A and 7B). Higher concentrations of mithramycin A (100 nM) led to reduced cell viability (data not shown). The decrease in ClC-2 with mithramycin A treatment supports the hypothesis that Sp1 and/or Sp3 are important regulators of basal ClC-2 expression.



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  		Figure 7. Mithramycin A reduces expression of ClC-2. PreII-19 cells, which express large amounts of endogenous ClC-2, were treated with 0, 25, and 75 nM of Mithramycin A for 72 h. Decreased expression of ClC-2 in cells treated with 25 nM or 75 nM Mithramycin is seen in A. Densitometric analysis of eight experiments (*P < 0.001) is shown in B.

 
Sp1 Isoforms Change with Development
Figure 8 demonstrates the expression patterns of Sp1 species in pCMV-Sp1 transfected and untransfected nuclear extracts and whole cell lysates. The adult L2 cells preferentially expressed the 105-kD species, whereas the 95-kD species predominated in pCMV-Sp1 transfected L2 cells. This switch in L2 cells from the 105-kD form to the 95-kD form and the concurrent increase in ClC-2 expression led us to question whether the 95-kD form is a more active species of Sp1. Support for this hypothesis resides in the preII-19 cells, which expressed high levels of ClC-2 and contained both 105-kD and 95-kD species of Sp1.



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  		Figure 8. Endogenous level of Sp1 varies between adult and fetal type II cell lines. A and B demonstrate Sp1 expression in 15-µg samples of whole cell lysates taken from transfected and untransfected L2 and preII-19 cell lines. The endogenous form of Sp1 found in untransfected L2 cells was 105 kD, whereas the preII-19 cell line contained both forms.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In mammalian lung, gene expression is highly regulated. Airway epithelial chloride conductance is critically important to airway fluid secretion in utero when lung differentiation is accelerated by increased fluid pressure (22–26). Fetal airways express multiple chloride conductances such as the cAMP-regulated CFTR, the ClC-2, and the calcium-activated chloride channels. High-level chloride secretion sustains a large secretory function of the fetal lung. This redundancy in chloride pathways may be protective in diseases in which one species malfunctions, such as in CF (27). This protection is lost after birth when the expression of many of these channels is downregulated. Elucidation of the specific mechanisms for regulation of these channels will help identify novel treatments to compensate for the functional deficits seen in CF.

Role of Sp1 and Sp3 in Perinatal Regulation of ClC-2 Expression
Sp1 and Sp3 both independently upregulated ClC-2 expression in adult rat lung type II cells. The rat ClC-2 promoter is enriched in GC boxes that bind Sp1 and Sp3 (1). Electrophoretic mobility shift assays with nuclear extract prepared from pre-type II cells confirmed the interaction of these transcription factors at sites within the first 237 bp of the promoter. We observed that overexpression of each factor independently upregulated rat ClC-2. Both Sp1 and Sp3 may be important in vivo, but these experiments suggest that there is a further redundancy in the system. To confirm that Sp1/Sp3 binding is critical to ClC-2 protein expression, mithramycin A, which blocks transcription factor binding to the GC promoter sequence, was observed to inhibit endogenous ClC-2 expression. Toxicity (cell death) was observed at 100 nM, suggesting that Sp1/Sp3 are critically important to cell viability. Indeed, the Sp1 knockout mouse, which dies at Day 11 of embryogenisis, and the Sp3 knockout mouse, which dies at birth of respiratory failure (18, 29), are supportive in vivo examples of the importance of these transcription factors to the developing organism.

Mechanism of Sp1 Mediation of Gene Expression
Sp1 exists in two isoforms that differ in degree of phosphorylation and glycosylation. Overexpression of Sp1 in fetal preII-19 cells produced both the 105-kD and 95-kD bands, whereas L2 cells produced predominantly the 105-kD band. The phosphorylation state of Sp1 has been described as an important factor for DNA binding (30–34), and phosphorylation of Sp1 via casein kinase II results in decreased DNA binding affinity. A review of our previous studies of Sp1 in rat lung homogenates (1) demonstrates that the lower molecular weight form of Sp1 is preferentially expressed in fetal lung tissue. In addition, the transfection of L2 cells led to an overexpression of the lower molecular weight form without significant change in the higher molecular weight form. The consequent increase in ClC-2 expression is most likely due to this lower molecular weight form. Though the understanding of the role of Sp3 is less clear, it is likely activated by a common regulatory pathway (29).

ClC-2 Is a Fetal Lung Chloride Channel
The exact role of ClC-2 in the developing lung is unclear. The ClC-2 knockout models as developed by Bosl and coworkers (35, 36) and by Nehrke and colleagues (36) have normal lungs by histopathology. Significant redundancy of chloride channels expressed in the developing lung allow the absence of a single chloride channel, such as ClC-2, to occur without major significance to fetal lung development. This appears to be the case for patients with CF, who lack CFTR and have normal lung development in utero. Interestingly, like the ClC-2 promoter, the CFTR promoter lacks a TATA box and has multiple Sp1 and Sp3 consensus sites (37). A perturbation in the expression of both channels via loss of transcription factors may lead to a disruption in this redundancy of chloride channels and perhaps significant disruption in pulmonary development.

This study augments the understanding of the mechanisms of ClC-2 expression and regulation. We have shown that Sp1 and Sp3 are critical for the developmental regulation and expression of ClC-2. In addition, expression levels and isoforms of Sp1 and likely Sp3 are developmentally regulated. Continued investigation is needed to define the interactions between Sp1, Sp3, and other genes critical to developing lung.


   Acknowledgments
 
The authors thank Dr. G. W. Hunninghake for the preII-19 cell line, Dr. Robert Tjian for the pCMV-Sp1, and Dr. Guntram Suke for the pCMV-Sp3. This work was supported by grants from the NHLBI (HL59410) and the Cystic Fibrosis Foundation (ZEITLI00G0) to P.L.Z. and (MOGAYZ01P0) to P.J.M. R.H. and M.J.M. were supported by student grants from the Cystic Fibrosis foundation (HALES01HO and MAXWEL02H0)

Received in original form January 28, 2003

Received in final form April 11, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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N. Vij and P. L. Zeitlin
Regulation of the ClC-2 Lung Epithelial Chloride Channel by Glycosylation of SP1
Am. J. Respir. Cell Mol. Biol., June 1, 2006; 34(6): 754 - 759.
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