Am J Physiol Lung Cell Mol Physiol 293: L453-L462, 2007.
First published June 8, 2007; doi:10.1152/ajplung.00084.2007
1040-0605/07 $8.00
Heterogeneity of transcription factor expression and regulation in human airway epithelial and smooth muscle cells
Alfredo Panebra,*
Mary Rose Schwarb,*
Clare B. Glinka, and
Stephen B. Liggett
Cardiopulmonary Genomics Program, University of Maryland School of Medicine, Baltimore, MD
Submitted 6 March 2007
; accepted in final form 4 June 2007
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ABSTRACT
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Transcription factors represent a major mechanism by which cells establish basal and conditional expression of proteins, the latter potentially being adaptive or maladaptive in disease. The complement of transcription factors in two major structural cells of the lung relevant to asthma, airway epithelial and smooth muscle cells, is not known. A plate-based platform using nuclear extracts from these cells was used to assess potential expression by binding to oligonucleotide consensus sequences representing >300 transcription factors. Four conditions were studied: basal, 
-agonist exposure, culture under proasthmatic conditions (IL-13, IL-4, TGF-, and leukotriene D4), and the dual setting of -agonist with proasthmatic culture. Airway epithelial cells expressed 70 transcription factors, whereas airway smooth muscle expressed 110. High levels of multiple transcription factors not previously recognized as being expressed in these cells were identified. Moreover, expression/ binding patterns under these conditions revealed extreme discordance in the direction and magnitude of change between the cell types. Singular (one cell type displayed regulation) and antithetic (both cell types underwent expression changes but in opposite directions) regulation dominated these patterns, with concomitant regulation in both cell types being rare (<10%). -Agonist evoked up- and downregulation of transcription factors, which was highly influenced by the proasthmatic condition, with little overlap of factors regulated by -agonists under both conditions. Together, these results reveal complex, cell type-dependent networks of transcription factors in human airway epithelium and smooth muscle that are dynamically regulated in unique ways by -agonists and inflammation. These factors may represent additional components in asthma pathophysiology or potential new drug targets.
transcription; -agonist; asthma; inflammation
IN EUKARYOTIC CELLS, TRANSCRIPTION factors represent the major elements by which gene transcription by RNA polymerase II is controlled (18, 26). Such factors regulate the basal expression of genes and serve to dynamically regulate gene expression during normal homeostatic conditions, exposures to xenobiotic agents such as therapeutic drugs or toxins, and pathological conditions, where they may be a basis for, or serve to counteract, aberrant cellular physiology (13, 18, 26). In asthma, two stromal cell types play significant roles in the syndrome: the airway epithelial cell and the airway smooth muscle cell (5, 9). Although some information is known about the regulation and action of selected transcription factors of immune cells of the lung and peripheral circulation (1, 11, 12, 24), there is a paucity of data on the expression of transcription factors in airway epithelial and smooth muscle cells. Furthermore, there are no studies that examine a large complement of transcription factors expressed in these cells and their modulation by inflammatory stimuli or -agonists, the most common treatment for asthma.
In the current study, we use a platform that quantitates the expression/binding capacity of transcription factors based on binding to 345 consensus oligonucleotide sequences. The two cell types were studied in the basal state, after exposure to a -agonist (a proasthmatic culture condition), and in the combination of both. We hypothesized that the complement of expressed transcription factors would be markedly different between airway epithelial and smooth muscle cells and that there would be significant differences in transcription-factor regulation under the above conditions between the two cell types. We indeed found extensive heterogeneity in the transcription factor expression/binding between airway epithelial and airway smooth muscle cells. This was found in terms of basal expression of individual factors, up- and downregulation by the aforementioned conditions, and marked cell-type dependency in the magnitude and direction of these changes.
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METHODS
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Cell culture.
BEAS-2B (R1 clone) cells were grown in Ham's F-12 medium (GIBCO) with 10% fetal calf serum, 5 µg/ml insulin, 5 µg/ml transferrin, 25 µg/ml epidermal growth factor, 100 U/ml penicillin, and 100 µg/ml streptomycin (14). Primary human airway smooth muscle cells were obtained from Clonetics (San Diego, CA) and were grown in monolayers in SmBM medium (Clonetics) (8). Both cell types were maintained at 37°C in a 95% air-5% CO2 atmosphere. Four treatment conditions were used, and each lasted 24 h at 37°C. Basal conditions consisted of no additives. Isoproterenol exposure was at a concentration of 10 µM. To mimic some aspects of a proasthmatic milieu, cells were incubated with 10 ng/ml IL-4, 10 ng/ml IL-13, 20 ng/ml TGF-, and 100 nM leukotriene D4. For simplicity, this is termed the "asthma condition." The fourth condition was concomitant treatments with isoproterenol and the aforementioned agents of the asthma condition. Nuclear extracts were prepared by using reagents from the NE-PER extraction system (Pierce, Rockford, IL) as previously described (19). Briefly, attached cells were placed on ice, and the medium was aspirated and washed three times with cold PBS. Cells were removed by gentle scraping, collected by centrifugation at 12,000 g for 15 min at 4°C, and swollen by incubation in cytoplasmic extraction reagent 1 buffer for 10 min. After incubation with the cytoplasmic extraction reagent 2 detergent solution for 1 min, preparations were centrifuged at 16,000 g for 5 min at 4°C. The nuclear pellet was resuspended in nuclear extraction buffer and was vortexed for 15 s every 10 min over 40 min, and the preparation was centrifuged at 16,000 g for 10 min at 4°C. The supernatant (nuclear extract) was collected, aliquotted, and frozen at –80°C until it was used. All of the above steps included the protease inhibitors leupeptin and aprotinin at 10 µg/ml.
Confocal microscopy.
To verify the cell type by characteristic histological features and antigen expression and to verify the homogeneity of the cells used for preparing nuclear extracts, confocal microscopy was carried out by using methods similar to those previously described (15). Briefly, cells were fixed with 4% paraformaldehyde and were permeabilized with 0.5% Triton X-100. The antibodies (Sigma) were vimentin conjugated to Cy3 (1:200) and cytokeratin conjugated to FITC (1:50), for identification of smooth muscle and epithelial cells, respectively. 4',6'-diamidino-2-phenylinodole dihydrochloride (DAPI, 0.1 µg/ml; Molecular Probes, Eugene, OR) was used as a fluorescent nuclear counterstain. Images were obtained on a Nikon Eclipse TE2000-E microscope at a magnification of x20.
Transcription factor expression arrays.
Nuclear extracts from the two cell types under the four conditions were assessed for the relative expression of 307 transcription factors by using the Panomics Combo Array (Panomics, Redwood City, CA) as described in detail elsewhere (10). The transcription factors that were quantitated in the array are found in Supplementary Table 1 (available online at The American Journal of Physiology-Lung Cellular and Molecular Physiology website). Standard abbreviations for the transcription factors, as found in the TRANSFAC (http://www.gene-regulation.com/pub/databases.html) or NCBI (http://www.ncbi.nlm.nih.gov) databases, are used in the tables and throughout the manuscript. Briefly, extracts were preincubated with biotin-labeled consensus oligonucleotides for the indicated transcription factors, the free probes were separated by washing, and the bound probes were extracted and hybridized to the array according to the manufacturer's protocol.
Horseradish peroxidase-based chemiluminescence was detected by using an AlphaImager (Alpha Innotech, San Leandro, CA) with black, white, and gamma values of 0, 65,500 and 0.55, respectively. The integrated density values were quantitated by using the AlphaEase software provided with the instrument. All plates were imaged under the same conditions.
In certain cases, when transcription factors are known to bind to more than one distinctly different position of a promoter, several oligonucleotide sequences were used in separate reactions with the nuclear extracts. The data from these are reported as the transcription factor name followed by a number in parentheses [i.e., "AP-2 (1)," "AP-2 (2)"]. Thus, although the array has 345 hybridization positions, these represent 307 transcription factors. For the analysis of changes in expression by the various conditions, each hybridization was considered a separate entity.
The lower limit of detection for the array is a density value of 10. Those factors with values <10 are reported as not detected. A relevant change in transcription factor expression was considered an increase or decrease of 50%. In cases where a condition induced expression from undetectable to >10, this is reported as upregulation, but a specific percentage of the change is not provided; similarly, detectable expression that changed to a value <10 is reported as downregulation without a notation as to the percent change. Comparisons were between isoproterenol vs. basal treatment, asthma culture conditions vs. basal, and asthma culture condition with isoproterenol vs. asthma condition alone. Data are presented as mean densities ± SD.
Miscellaneous.
-Adrenergic receptor (-AR) expression was determined with cell membranes by using 125I-cyanopindolol (125I-CYP) binding as previously described (6, 7). Because neither cell type expresses the 1-AR subtype, the specific binding from these assays represents the 2-AR subtype. Protein concentrations of nuclear extract and cell-membrane preparations were quantitated by the copper bicinchoninic acid method (23).
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RESULTS
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Figures 1 and 2 are representative confocal-microscopy images of the cells used in the current study. As shown, they have the characteristic morphological appearance of epithelial and smooth muscle cells under white light (Fig. 1, A and D, and Fig. 2, A and D). Nuclei are indicated by the blue counterstain from DAPI (Fig. 1, B and E, and Fig. 2, B and E). The epithelial cells revealed intense images from incubation with the antibody to cytokeratin (Fig. 1C) but not to vimentin (Fig. 2C). In contrast, airway smooth muscle revealed intense signals from vimentin and none from cytokeratin (Fig. 2F and Fig. 1F, respectively). As expected, both cell types expressed 2-ARs. With the use of 125I-CYP radioligand binding, these were quantitated at 213 ± 8.4 and 78 ± 12 fmol/mg, respectively, for airway epithelial cells and airway smooth muscle cells (n = 6).
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Fig. 1. Confocal microscopy of airway epithelial (AEC) and airway smooth muscle (ASM) cultured cells used for transcription factor-expression studies. Cells were prepared as described in METHODS. Morphological characteristics (A and D) are consistent with cell type as is the cytokeratin-positive staining found only with AEC (C and F). Signals in B and E are from fluorescent nuclear counterstain 4',6'-diamidino-2-phenylinodole dihydrochloride (DAPI). Magnification, x20.
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Fig. 2. Confocal microscopy of AEC and ASM cultured cells used for transcription factor-expression studies. Cells were prepared as described in METHODS. Morphological characteristics (A and D) are consistent with cell type as is the vimentin-positive staining found only with ASM (C and F). Signals in B and E are from fluorescent nuclear counterstain DAPI. Magnification, x20.
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Tables 1 and 2 show the results of the transcription factor-binding assays from the two cell types, where signals were above the level of detection for any one of the four conditions. The method used not only requires expression of the factor but also any posttranslational modifications, such as phosphorylation or other conditions necessary for binding to the consensus oligonucleotide. This may provide a particular advantage in assessing the regulation of transcription factor activity under relatively short-term conditions. In the intact cell, this would represent expression of a "functional factor" capable of altering transcription through such binding. In this study, for simplicity, we refer to this output as representing transcription-factor "expression" or "expression/binding," recognizing that there may be additional aspects that ultimately result in the signal. The complement of transcription factor binding sites queried by these arrays is provided in Supplemental Table 1. For the airway epithelial cells, expression of 70 transcription factors was noted, whereas 110 factors were detected in airway smooth muscle cells (Fig. 3). In epithelial cells, 10 (
15%) of the transcription factors were unique to these cells compared with airway smooth muscle (Table 3). In the latter, 49 transcription factors (45%) were found to be unique. The two cell types had common expression of 55 transcription factors (Table 3).
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Fig. 3. Global expression of transcription factors in AEC and ASM based on a query of >300 factors. Shown is number of transcription factors detected by consensus oligonucleotide binding under any of 4 culture conditions (see METHODS). Factors expressed only in 1 cell type are denoted as unique and those detected in both as common.
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The four conditions resulted in multiple changes in transcription factor expression/binding in both airway epithelial and smooth muscle cells (Fig. 4). For airway epithelial cells, at baseline 50 factors were expressed, and 16 were increased with isoproterenol. For five of these [E2F-2, E4F, EGR, PARP, and Pax-4a], isoproterenol resulted in an increase in expression/binding over detectable basal levels. At baseline, 10 factors were not detected but were induced by isoproterenol exposure. -Agonist exposure reduced expression/binding of 10 factors. In the asthma-culture setting, 12 factors were increased over baseline; of these, 10 were undetectable at baseline. Four transcription factors were decreased from baseline under these conditions (Fig. 4A). Some studies have shown -agonists to have anti-inflammatory properties, so for purposes of data presentation the effect of isoproterenol on the transcription factor binding while in the asthmatic culture conditions was used for comparison (i.e., relationship between transcription factor expression in the asthma culture conditions alone vs. expression under the combined condition). Here we found 18 factors that were increased by isoproterenol exposure under the asthma-culture conditions (Fig. 4A). Of these, 11 were not detectable in the asthma condition alone. Of the 34 (total) factors increased by isoproterenol exposure under either of the two conditions, there was overlap of only six factors [AP-2, Elk-1, E4F, Evi-1, PEBP2
, and PARP] that were increased by isoproterenol alone and isoproterenol in the asthma-culture setting. Sixteen factors were decreased by isoproterenol compared with the asthma plus isoproterenol conditions. Six of these (NF-Y, RAR, T3R, USF, CTCF, and HOX D9,10) were also decreased in the isoproterenol-alone setting.
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Fig. 4. Conditional changes in transcription factor expression and/or binding in AEC and ASM. Cells were treated with -agonist isoproterenol (iso), a proasthma cocktail (see METHODS), or both for 24 h, and nuclear extracts were prepared. y-Axis represents number of transcription factors that changed by condition, with number of upregulated factors in closed bars and number of downregulated factors in open bars.
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For airway smooth muscle cells, a significantly greater number of transcription factors were detected under at least one of the four conditions compared with airway epithelial cells. For these cells, expression/binding of 110 transcription factors was found, with 96 being expressed under basal conditions. Isoproterenol exposure resulted in an increase in 19 transcription factors, of which 13 were undetectable at baseline (Fig. 4B; Table 2). Sixteen factors were decreased by isoproterenol. Under the asthma-culture conditions, 8 factors were increased (7 were not detected at baseline) and 14 were decreased. Expression changes evoked in the isoproterenol-with-asthma culture conditions compared with the asthma condition alone amounted to 32 factors that were upregulated and 10 that were downregulated (Fig. 4B). Of these 42 total factors that were regulated under the dual condition, 11 also underwent changes in the same direction during -agonist treatment alone (8 increased, 3 decreased).
As introduced earlier, there were 55 factors that were expressed in both cell types, and we considered whether these were regulated in a similar manner under the various conditions. We found evidence for regulation that could be classified in three categories (Fig. 5). A singular change denotes that there was a change in expression/binding in either direction but it was found in only one cell type. Concomitant regulation is defined as a change in expression/binding in the same direction (up- or downregulation) that is observed in both cell types under a given condition. Antithetic regulation is a change in expression/binding of a transcription factor under a given condition, but the direction of the change is opposite between airway epithelial cells and smooth muscle cells. For the current conditions, we found that singular changes dominated in this group of transcription factors that were expressed in both cell types. Of particular interest were the antithetic changes (Fig. 5, red:blue or blue:red), which were observed in 60% of cases where changes occurred with both cell types. These factors were AP-2, NF-E1, E4F (observed twice), Elk-1, COUP-TF; observed twice, EGR1, and PU.1. Surprisingly, in all but one case of antithetic regulation, expression of the given factor increased in the airway epithelial cells and decreased in the airway smooth muscle cells. Concomitant regulation was less frequent, with no specific pattern as to a predominance of up- or downregulation. Although airway epithelial cells express greater than twofold more 2-ARs compared with the smooth muscle cells, there was no trend toward a greater number of -agonist-regulated transcription factors (either as an absolute number or percentage of total transcription factors) in these cells vs. smooth muscle.
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Fig. 5. Regulation of transcription factors that are expressed in both AEC and ASM. Shown are transcription factors where expression was common to both cell types under at least 1 of 4 conditions. Red bars indicate an increase and blue bars a decrease in expression/binding by indicated condition. Yellow bars represent the scenario where expression was detectable, but there was no change with condition. White bars indicate that there was no detectable expression for the transcription factor under given condition.
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DISCUSSION
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Transcription factor binding to specific sequences in regulatory regions is now recognized as a critical component in establishing gene expression under basal, conditional, and cell type-dependent conditions, and transcription factors may be particularly amenable as drug targets in the lung for treatment of asthma and chronic obstructive lung disease (2, 3, 16, 21). As a first step in understanding their role in airway epithelial and smooth muscle cells, we undertook analysis of potential expression/binding of >300 transcription factors and present this data for other investigators to use for specific gene-transcription issues. Our global analysis of the data reveals several important findings. There were substantially more transcription factors expressed in airway smooth muscle cells compared with epithelial cells, and of the epithelial cell factors, less than half were in common with those detected in smooth muscle cells. This underlies the important concept that whereas cells may express many common proteins, the mechanism(s) by which basal and/or regulated expression is achieved at the transcriptional level may be quite different.
We also show that exposure to -agonist, a common treatment for obstructive lung disease, alters the binding levels of 23% of the expressed transcription factors in epithelial cells and 38% in airway smooth muscle cells. The impact of these changes is yet to be explored, but it is likely that there are those that are detrimental in terms of the cellular or organ physiology in these two syndromes. Indeed, -agonist use continues to be controversial in asthma, with multiple reports of increased mortality or other indices of poor asthma control during continuous (chronic) therapy (4, 17, 22, 25). Given the efficacy of -agonists for relieving bronchospasm, one strategy for new, combined, pharmacological therapy may involve an additional agent that inhibits dysregulation of certain transcription factors by -agonists. The therapeutic effect of glucocorticoids in asthma may indeed be in part due to this mechanism (3).
The role of transcription factors in immune cells has revealed a number of important pathways that initiate or maintain inflammation (1, 3, 11, 12, 16, 24). Here we show that these two resident structural cells, airway epithelial and smooth muscle, also undergo changes in expression of transcription factors in an inflammatory environment. These may provide the link, or the "amplification," necessary for the proasthmatic signals that reach these cells to evoke pathological sequelae. These would include the altered mucous content and production from epithelial cells, increased smooth muscle mass, hyperresponsiveness to contraction by smooth muscle, and resistance to -agonist-promoted relaxation. As such, transcription factor "agonists" and "antagonists," inhibitors of protein kinases that phosphorylate transcription factors, and chromatin-modifying agents have been proposed as therapeutic agents for asthma (20).
It is also intriguing to consider the effect of -agonist in the context of the asthmatic-culture conditions. In airway epithelial cells, multiple transcription factors displayed increased and decreased expression/binding under these conditions. However, of the 43 factors that underwent expression changes under the dual condition, only 11 of these displayed the same changes with -agonist alone. In airway smooth muscle, about three times more transcription factors were upregulated under the dual conditions compared with -agonist exposure alone, and again, there was little overlap in the factors that underwent up- or downregulation between the two conditions. This clearly adds an additional layer of complexity in terms of new pathway and drug discovery, in that the advantageous or deleterious effects of -agonists on transcription factor expression in these cells may be dependent on the severity and characteristics of the inflammation. Our findings with the transcription factors that are in common with the two cell types also raise some intriguing issues. Of particular note is the heterogeneity of the directional changes by cell type. There were multiple instances where a factor from one cell type was up- or downregulated by a given condition with no change, or the opposite change, observed in the other cell type. In fact, concomitant regulation in the same direction in both cell types was rare. This further highlights the cell-type specificity of the regulation of transcription factors and cautions against overgeneralization about such processes when studying a single cell type.
In summary, we have identified transcription factor expression/binding in human airway epithelial and smooth muscle cells under basal conditions, as well as three other conditions relevant to asthma. We found substantial differences in the complement of transcription factors expressed between the two cell types. In addition, -agonist exposure, a proasthmatic inflammatory milieu, and the combination of both conditions evoked distinct regulatory events that differed by transcription factor and the direction and magnitude of the change between the cell types. The identification of these transcription factors, and their regulation, provides additional insight into potential adaptive and maladaptive events in asthma and may represent novel therapeutic targets.
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GRANTS
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This work was funded by National Heart, Lung, and Blood Institute Grants HL-45967, HL-71609, and HL-65899.
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ACKNOWLEDGMENTS
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We thank Esther Moses for manuscript preparation.
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FOOTNOTES
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Address for reprint requests and other correspondence: S. B. Liggett, 20 Penn St., HSF-II, Rm. S-114, Baltimore, MD 21201-1075 (e-mail: sligg001{at}umaryland.edu)
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. Section 1734 solely to indicate this fact.
* A. Panebra and M. Schwarb contributed equally to this work. 
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Copyright © 2007 by the American Physiological Society.
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ABSTRACT
METHODS
