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. Author manuscript; available in PMC: 2026 Jul 28.
Published in final edited form as: J Cell Physiol. 2015 Feb;230(2):464–472. doi: 10.1002/jcp.24729

C/EBPα and the Vitamin D Receptor Cooperate in the Regulation of Cathelicidin in Lung Epithelial Cells

PUNEET DHAWAN 1, RAN WEI 1, CHENG SUN 1, ADRIAN F GOMBART 2, H PHILLIP KOEFFLER 3, GILL DIAMOND 4, SYLVIA CHRISTAKOS 1,*
PMCID: PMC13404115  NIHMSID: NIHMS2190985  PMID: 25078430

Abstract

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) and the vitamin D receptor (VDR) have been reported to have an important role in the regulation of innate immunity. We earlier reported that the antimicrobial peptide cathelicidin is induced by 1,25(OH)2D3 in normal human bronchial epithelial cells with a resultant increase in antimicrobial activity against airway pathogens. In this study, we demonstrate that C/EBP alpha (C/EBPα) is a potent enhancer of human cathelicidin antimicrobial peptide (CAMP) gene transcription in human lung epithelial cells. In addition we found that C/EBPα functionally cooperates with VDR in the regulation of CAMP transcription. A C/EBP binding site was identified at −627/−619 within the CAMP promoter, adjacent to the vitamin D response element (VDRE; −615/−600). Mutation of this site markedly attenuated the transcriptional response to C/EBPα as well as to 1,25(OH)2D3, further indicating cooperation between these two factors in the regulation of CAMP. ChIP analysis using 1,25(OH)2D3 treated human lung epithelial cells showed C/EBPα and VDR binding to the CAMP promoter. C/EBPα has previously been reported to cooperate with Brahma (Brm), an ATPase that is component of the SWI/SNF chromatin remodeling complex. We found that dominant negative Brm significantly inhibited C/EBPα as well as 1,25(OH)2D3 mediated induction of CAMP transcription, suggesting the functional involvement of Brm. These findings define novel mechanisms involving C/EBPα, SWI/SNF, and 1,25(OH)2D3 in the regulation of CAMP in lung epithelial cells. These mechanisms of enhanced activation of the CAMP gene in lung epithelial cells suggest potential candidates for the development of modulators of innate immune responses for adjunct therapy in the treatment of airway infections.

The respiratory epithelium is exposed to a large number of potentially pathogenic microorganisms. A principal defense mechanism protecting the lungs against infection is the production of antimicrobial peptides including cathelicidins (Singh et al., 2000; Laube et al., 2006). Since bacteria do not develop resistance to antimicrobial peptides, there is increased interest in these antimicrobial agents due to their potential as therapy against antibiotic resistant pathogens (Ahmad et al., 2012). Cathelicidins are a family of proteins that originate from a precursor molecule that contains a conserved N-terminal and a less conserved C-terminal cationic antimicrobial peptide domain that is activated by proteolytic cleavage from the N-terminal cathelin segment of the propeptide (Lai and Gallo, 2009). The only known human cathelicidin is LL-37, the C-terminal domain of human cationic antimicrobial protein 18 (hCAP-18) (Vandamme et al., 2012). It is encoded by the human cathelicidin antimicrobial peptide (CAMP) gene. CAMP was first identified in specific granules of neutrophils and was subsequently identified in monocytes and macrophages, dendritic cells, lymphocytes, mesenchymal stem cells, bone marrow stroma and epithelial cells of the skin, gastrointestinal tract and respiratory tract. CAMP has broad spectrum activity against Gram positive and Gram negative microorganisms (Vandamme et al., 2012). CAMP has also been to found to act as a chemokine, to modulate dendritic cell maturation, to promote wound healing and to stimulate angiogenesis (Vandamme et al., 2012). These findings suggest that CAMP, through multiple functions, plays a critical role in host defense.

Due to the increased prevalence of antibiotic resistant pathogens, one approach to fight bacterial infections is to induce endogenous expression of antimicrobial peptides for therapeutic benefit (Ahmad et al., 2012). Although 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) was shown to be a potent inducer of CAMP in myeloid cells and in keratinocytes (Wang et al., 2004; Gombart et al., 2005; Weber et al., 2005), the effects of 1,25(OH)2D3 in airway epithelial cells had not been examined. We previously demonstrated that CAMP mRNA and hCAP18 are induced by 1,25(OH)2D3 in human bronchial epithelial cells and that airway surface fluid from 1,25 (OH)2D3 treated cells exhibits increased antimicrobial activity, indicating CAMP up-regulation correlated with antibacterial activity induction (Yim et al., 2007). Vitamin D is known to have an important role in calcium homeostasis and bone development and maintenance. However, recent evidence provides evidence for an interrelationship between vitamin D and health beyond bone, including effects on immune function (Christakos and DeLuca, 2011). Epidemiological studies have indicated that vitamin D deficiency predisposes humans to respiratory tract infection and that vitamin D sufficiency is associated with increased resistance to these infections (Hansdottir and Monick, 2011). However the mechanisms are not understood. The actions of 1,25(OH)2D3 are mediated, similar to other steroids, by the nuclear receptor (VDR) which heterodimerizes with the retinoid × receptor and interacts with DNA sequences (vitamin D response elements) in target genes (Christakos, 2008; Pike and Meyer, 2010). It has been suggested that cell and promoter specific functions of VDR are mediated through differential recruitment of coactivators and that increased interaction between VDR and coactivators may be a major mechanism the couples extracellular signals to vitamin D action (Christakos, 2008; Pike and Meyer, 2010). Understanding tissue specific factors that regulate CAMP gene expression may be the key to therapeutic manipulation of endogenous CAMP expression.

In this study we provide evidence that C/EBPα, which is induced by 1,25(OH)2D3 in lung epithelial cells, is a potent enhancer of CAMP transcription and that C/EBPα cooperates with VDR and Brahma (Brm) (an ATPase that is a component of the SWI/SNF chromatin remodeling complex) in the regulation of CAMP transcription. Our findings define novel mechanisms involving C/EBPα, the SWI/SNF complex and 1,25(OH)2D3 in the regulation of CAMP gene expression in lung epithelial cells and suggest candidates for the development of modulators of innate immune responses to airway infection.

Experimental Procedures

Materials

[α−32P]ATP (3,000 Ci [111 TBq]/mmol) was purchased from NEN Life Science Products (Boston, MA). The polyvinylidene difluoride (PVDF) membranes and the pre-stained protein molecular weight markers were purchased from Bio-Rad Laboratories, Inc. (Hercules, CA). Primary antibodies against C/EBPα VDR, Brm, and β-actin, and secondary antibodies against mouse and rabbit antisera were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). 1,25(OH)2D3 was purchased from Cayman Chemicals (Ann Arbor, MI).

Cell culture

Cell culture media DMEM and 0.25% trypsin-EDTA and penicillin-streptomycin-neomycin (PSN) antibiotic mixture were purchased from Life Technologies (Carlsbad, CA). A549 adenocarcinomic human alveolar basal epithelial cells and BEAS-2B human bronchial epithelial cells were obtained from American Type Culture Collection (Manassas, VA). Normal human bronchial epithelial (NHBE) cells were obtained from Lonza, Inc. (Walkersville, MD). Fetal bovine serum (FBS) and charcoal-stripped FBS were from Gemini Biological Products (Calabasas, CA). A549 cells and BEAS-2B cells were cultured in DMEM supplemented with 10% heat-inactivated FBS and 1% antibiotic mixture PSN. NHBE cells were cultured in BEGM (Bronchial Epithelial Cell Growth Medium) supplemented with Growth Factors (Lonza, Inc., Walkersville, MD). Cells were grown in a humidified incubator with atmosphere of 95% air-5% CO2 at 37 °C. For treatments, cells were grown to desired confluence and their medium was changed to medium supplemented with 2% charcoal-dextran-treated FBS. Treatments with vehicle or 1,25(OH)2D3 were done for the durations and with concentrations described in the figure legends.

Plasmids, transfections and assay of luciferase activity

The luciferase reporter construct of human CAMP promoter (−693 to +17) was generated as previously described (Gombart et al., 2005). The C/EBP site at −627/−619 was mutated by site directed mutagenesis using site directed mutagenesis kit from Stratagene. The oligonucleotides used to generate the mutated C/EBPα site (C/EBP site shown in bold, mutated nucleotides underlined) were as follows: 5’-AACTGCAACTTACTCTTCCCGGGTTCAATGG-3’ and 5’-CCATTGAACCCGGGAAGAGTAAGTTGCAGTT-3’. The wild type and the mutant promoter constructs were used for reporter assays in A549 human alveolar basal epithelial cells and BEAS-2B human bronchial epithelial cells. Due to low transfection efficiency, NHBE cells were not used for studies examining transcriptional regulation of CAMP using transfected cells. The C/EBPα, β, and δ expression vectors were a gift of Simon Williams, Texas Tech University (Lubbock, TX). The dominant negative (DN) C/EBP construct was a gift from Charles Vinson (National Cancer Institute, Bethesda, MD). pCMV-Brm and pCMV-mutant Brm (with the ATPase site mutated that acts as dominant negative inhibitor) were obtained from Moshe Yaniv (Institut Pasteur, Paris, France) (Muchardt et al., 1996). pAV-hVDR was a gift of J. Wesley Pike (University of Wisconsin, Madison). Since low levels of VDR were previously reported in A549 cells (Kim et al., 2012), VDR was cotransfected in studies examining the effect of 1,25 (OH)2D3 on CAMP transcription. Empty vectors were transfected to keep the total DNA concentration equal. Cells were transfected using Lipofectamine 2000 (Invitrogen) treated as described in Results in the appropriate medium supplemented with 2% charcoal-dextran-treated FBS. After treatment with vehicle or the compounds noted at the concentrations and times indicated in Results, cells were harvested and a dual luciferase assay was performed according to the manufacturer’s protocol (Promega, Madison, WI).

Electrophoretic mobility shift assay (EMSA)

Complementary oligonucleotides containing either the wild-type (forward 5’-AACTGCAACTTCTGCTTCCCGGGTT-CAATGG-3’ and reverse oligonucleotide 5’-CCATTGAA-CCCGGGAAGCAGAAGTTGCAGTT-3’) or the mutant C/EBP site (forward 5’-AACTGCAACTTACTCTTCC-CGGGTTCAATGG-3’ and reverse 5’-CCATTGAACCCGG-GAAGAGTAAGTTGCAGTT-3’) were synthesized by the Rutgers-New Jersey Medical School Molecular Resource Facility, Newark, New Jersey. The complementary oligos were annealed, 5’ end-labeled with 32P-ATP, purified and used for electrophoretic mobility shift assay (EMSA) as described previously (Dhawan et al., 2005). Briefly, 5 μg of the nuclear extracts from C/EBPα-transfected cells were incubated for 20 min at 25 °C with 2 μg of poly (dI/dC) with or without unlabeled specific or nonspecific DNA competitor or C/EBPα antibody in binding buffer (4 mM Tris-HCl [pH 7.9], 1 mM EDTA [pH 8.0], 60 mM KCl, 12% glycerol, 12 mM HEPES, and 1 mM dithiothreitol). This was further incubated with 0.5 ng of the labeled oligonucleotide probe (approx. 100,000 cpm) for 30 min at 25 °C. The samples were electrophoresed for 2.5 h at 4 °C on a 6% nondenaturing polyacrylamide gel that had been pre-electrophoresed for 30 min at 100 V/cm at 4 °C in 45 mM Tris-45 mM boric acid-1 mM EDTA. The gel was dried and exposed to X-ray film at −80 °C with intensifying screens.

Nuclear extracts

Nuclear extracts were prepared using the NE-PER nuclear and cytoplasmic extraction reagent kit (Thermo Scientific, Rockford, IL) as per manufacturer’s protocol. Protease inhibitor cocktail “Complete Mini” was also used during the process (Roche Diagnostics, Indianapolis, IN). The protein concentration of the nuclear extracts was measured by the Bradford assay (Bradford, 1976), and aliquots were stored at −80 °C.

Messenger RNA analysis

RNA-Bee reagent (Amsbio, Cambridge, MA) was used to isolate total RNA from cultured cells. For semi quantitative RT-PCR analysis of C/EBPα and VDR mRNA levels, RT-PCR was performed using 4 μg total RNA and Superscript III Reverse transcriptase with Ampli Taq gold DNA polymerase (Life Technologies). Primers used were as follows: C/EBPα forward 5’CGGTGGACAAGAACAGCAAC-3’ and reverse, 5’CGG-AATCTCCTAGTCCTGGC-3’ (35 cycles); VDR forward 5’ ATCTGCATCGTCTCCCCAGAT-3’ and reverse, 5’AGCG-GATGTACGTCTGCAGTG-3’ (35 cycles); hCAMP forward 5’-GCTAACCTCTACCGCCTCCT-3’ and reverse 5’-GGTCACTGTCCCCATACACC (38 cycles); β-actin forward 5’-CCTGTGGATCTGACAGCTGAA-3’ (35 cycles) and reverse 5’-TCCCAAATCGGTTGGAGATA-3’ (35 cycles); GAPDH forward 5’-TCACCATCTTCCAGGAGCG-3’ and reverse 5’-CTGCTTCACCACCTTCTTGA-3’ (35 cycles). The cycles were chosen so that the amplification was conducted in the linear range of amplification efficiency. The resulting PCR products were subjected to electrophoresis on a 1% agarose gel containing ethidium bromide and bands were visualized under UV light. Gel data were recorded using the Gene Genius bioImaging System (Syngene, Frederick MD) and relative densities of the bands were determined using ImageJ software (NIH, Bethesda, MD). Data were normalized for the expression of β-actin mRNA within the sample. Real time quantitative PCR (RTQ-PCR) was also used to quantify CAMP mRNA in NHBE cells using MyCycler (Bio-Rad Laboratories, Hercules, CA) using 2× SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) in a volume of 20 μl. RTQ-PCR primers used were: hCAMPforward, 5’-GTCACCAGAGGATTGTGACTTCAA-3’ and reverse, 5’-TTGAGGGTCACTGTCCCCATA-3’; β actin forward, 5’-AGTCTGTGGCATCCACGAAACTAC-3’, and reverse, 5’-CTTCTGCATCCTGTCGGCAATG-3’. The PCR fragments were amplified for 45 cycles (15 sec at 95 °C and 1 min at 60 °C which provided optimal melt curves for all primer pairs). The relative CAMP mRNA expression in response to 1,25 (OH)2D3 was calculated using the 2−ΔΔCt method, normalized to β-actin and presented as fold induction.

Chromatin immunoprecipitation (ChIP) assay

A549 cells or NHBE cells, treated with vehicle or 1,25(OH)2D3 were used for the Chromatin immunoprecipitation (ChIP) assays as previously described (Shen and Christakos, 2005; Dhawan and Christakos, 2010). In brief, treated cells were washed with PBS and cross-linked using 1% formaldehyde for 15 min. Crosslinking was stopped by adding glycine to a final concentration of 0.125 M. The cells were washed twice with ice-cold PBS and were collected by scraping. The cells were lysed for 20 min each, first in buffer 1 (5 mM Pipes, pH 8.0, 85 mM KCl, 0.5% Nonidet P-40) followed by buffer 2 (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.1). The resulting chromatin pellet was sonicated to an average DNA size of 500 bp (evaluated by 1% agarose gel electrophoresis) using a Fisher model 100 sonic dismembranator at a power setting of two. The sonicated extract was centrifuged for 10 min at 13,000 rpm at 4 °C and then diluted into ChIP dilution buffer (16.7 mM Tris-HCl, pH 8.1, 150 mM NaCl, 0.01% SDS, 1.1% Triton X-100, and 1.2 mM EDTA). Immunoprecipitations were performed at 4 °C overnight with the indicated antibody. After 4 h incubation with protein A agarose (Rockland Immunochemicals, Inc., Gilbertsville, PA), the beads were collected by centrifugation. The protein A agarose beads were washed sequentially as previously described (Shen and Christakos, 2005; Dhawan and Christakos, 2010). The protein-DNA was then eluted by using 1% SDS and 0.1 M NaHCO3 for 15 min twice. Cross-links were reversed by incubating at 65 °C overnight in elution buffer with 0.2 M NaCl. DNA fragments were purified using the Qiagen QIAquick PCR purification kit (Valencia, CA) and PCR was performed using primers designed to amplify fragments of human CAMP promoter containing the C/EBP and VDR binding sites (forward, 5’GTT ACC CAG GCT GGA GTG C 3’; reverse, 5’ ACG GTC TGC ACG CCT ATA AT −3’). PCR analysis was performed in the linear range of DNA amplification. 250 bp PCR products were resolved in 1% agarose gel and visualized using ethidium bromide staining. DNA obtained before precipitation was used as the input. 10% of input was used for PCR reaction. DNA acquired from immunoprecipitates performed with IgG was subjected to PCR using the primers designed to amplify the fragment containing the C/EBP/VDR binding site to exclude nonspecific binding.

Re-ChIP experiments were also performed using sequential chromatin immunoprecipitation and two different antibodies (α-C/EBPα and α-Brm) to assay for the simultaneous presence of these two factors at the same site in the CAMP promoter. In Re-ChIP experiments, on the second day of the ChIP experiment, complexes were eluted in 60 ml of elution buffer containing 10 mM dithiothreitol for 30 min at 37 °C. The eluted samples were diluted 50 times with ChIP dilution buffer and subjected again to the ChIP procedure using the second antibody (α-Brm).

Statistical analysis

Results are expressed as means ± standard errors (SE), and significance was determined by analysis by Student’s t-test for two-group comparison or by analysis of variance for multiple-group comparison.

Results

VDR and C/EBPα cooperate in the regulation of CAMP transcription in lung epithelial cells

Although 1,25(OH)2D3 induces antimicrobial peptides in myeloid cells and keratinocytes, very little is known about the effect of 1,25(OH)2D3 on antimicrobial activity and the mechanisms by which 1,25(OH)2D3 modulates innate immunity in airway epithelial cells. We previously reported the induction of CAMP by 1,25(OH)2D3 in NHBE cells and the resultant increase in antimicrobial activity against airway pathogens (Yim et al., 2007; Fig. 1A). To examine the mechanism of activation of CAMP gene expression by 1,25 (OH)2D3, A549 human pulmonary epithelial cells were transfected with the CAMP promoter (−693/+17) luciferase construct and treated with 1,25(OH)2D3. A concentration dependent induction of transcriptional activity was observed (2- and 7-fold induction in response to 10 and 100 nM 1,25 (OH)2D3 treatment for 24 h, respectively) (Fig. 1B). Because sequence analysis indicated putative C/EBP binding sites in the CAMP promoter, we investigated the possibility that C/EBPs may be involved in the regulation of CAMP transcription. C/EBPα (0.025–0.25 μg) significantly enhanced CAMP transcription (3.6–8.4 fold; P < 0.05 compared to vector transfected control) (Fig. 1C). Although 1,25(OH)2D3 (10 nM) or C/EBPα (0.1 μg) stimulates CAMP transcription 2-, 3- and 6-fold, respectively, C/EBPα transfection in A549 cells combined with 1,25(OH)2D3 treatment resulted in a 13 fold induction of CAMP transcription, suggesting functional cooperation between C/EBPα and 1,25(OH)2D3 in the regulation of CAMP transcription (Fig. 1D, left panel). Unlike C/EBPα, C/EBPβ did not significantly affect CAMP transcription (Fig. 1D, left panel). C/EBPδ was also not found to stimulate CAMP transcription (not shown). Similar results were observed using BEAS-2B cells (Fig. 1D, right panel). In addition, RT-PCR analysis indicated that CAMP gene expression was induced by 1,25(OH)2D3, C/EBPα and the combination of C/EBPα and 1,25(OH)2D3 in A549 cells (Fig. 1E, left panel) and in BEAS-2B cells (Fig. 1E, right panel), consistent with a role of C/EBPα alone and in cooperation with VDR in the regulation of CAMP gene expression.

Fig. 1.

Fig. 1.

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Functional cooperation between VDR and C/EBPα in the regulation of CAMP transcription in human lung epithelial cells. (A) RTQ-PCR was performed using total RNA from NHBE cells treated with vehicle or 1,25(OH)2D3 (10 nM for 6 and 24 h). *P < 0.05 compared to zero time. Lower concentrations of 1,25(OH)2D3 (1 nM and below for 6 and 24 h) fail to elicit a response; Yim et al. (2007). (B) 1,25(OH)2D3 induces CAMP transcription in human pulmonary epithelial cells. Cells were transfected with the CAMP promoter (–693/+17) (250 ng) and VDR expression plasmid (50 ng) and treated with 1,25(OH)2D3 (1 nM to 100 nM) for 24 h. CAMP promoter activity is represented as fold induction (mean ± SE; n = at least three separate experiments) by comparison to basal levels. *P < 0.05 compared to cells treated with vehicle (Basal). (C) A549 cells were co-transfected with the CAMP promoter (−693/+ 17) and increasing concentrations of C/EBPα. *P < 0.05 compared to basal. (D) C/EBPα induces CAMP transcription and cooperates with 1,25(OH)2D3 in the regulation of CAMP transcription. Top panel: C/EBPα promoter schematic showing the location of the VDRE (−615/−600) and the putative C/EBP binding site (−627/−619). Left panel: A549 cells were transfected with the CAMP promoter (–693/+ 17) (250 ng), VDR (50 ng) and C/EBPα (0.1 μg) or C/EBPβ (0.1 μg) expression plasmids. Empty vectors were used to keep the total DNA concentration the same. Transfected cells were treated with vehicle or 1,25(OH)2D3 (10 nM) for 24 h. CAMP promoter activity is expressed as fold induction (mean ± SE; n = 6–8 separate experiments) by comparison to basal levels. Right panel: Induction of CAMP transcription in BEAS-2B cells was determined as described for A549 cells. CAMP promoter activity is expressed as fold induction (mean ± SE; n = 3–4 separate experiments) by comparison to basal. *P < 0.05 compared to basal. The induction of CAMP promoter activity by the combination of 1,25(OH)2D3 and C/EBPα is significantly greater than CAMP promoter activity in response to C/EBPα alone or 1,25(OH)2D3 alone, + P < 0.01. (E) Representative RT-PCR analysis for CAMP mRNA expression in A549 cells (left panel) and BEAS-2B cells (right panel) treated as described in 2D.

C/EBPα expression is induced by 1,25(OH)2D3 in human lung epithelial cells

Since we previously reported the regulation of C/EBP family member by 1,25(OH)2D3 in different cell types,weexamined the possibility that C/EBPα is a 1,25(OH)2D3 target in lung epithelial cells. When A549 cells were treated with increasing concentrations of 1,25(OH)2D3 Western blot analysis using nuclear extracts from vehicle (Basal) or 1,25(OH)2D3 treated cells showed a significant induction of C/EBPα expression at 1, 10, and 100 nM 1,25(OH)2D3 (Fig. 2A). The induction of C/EBPα in A549 cells was accompanied by an increase in the expression of VDR (Fig. 2A). C/EBPα and VDR were also significantly induced by 1,25(OH)2D3 (10 nM 1,25(OH)2D3 treatment for 24 h) in NHBE cells (Fig. 2B). Induction of C/EBPα and VDR mRNA by 1,25(OH)2D3 in NHBE cells is shown in Fig. 2C.

Fig. 2.

Fig. 2.

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C/EBPα and VDR are induced by 1,25(OH)2D3 in lung epithelial cells. (A) 1,25(OH)2D3 dose response in A549 cells. Top panel: Representative Western blot. Western blot analysis was performed using nuclear extracts from A549 cells treated with vehicle or 1,25 (OH)2D3 (1 nM to 100 nM) for 24 h and probed with C/EBPα, VDR and β-actin antibodies. Lower Panel: Graphic representation of densitometric scans of Western blots. C/EBPα and VDR are significantly induced by 1, 10, and 100 nM 1,25(OH)2D3 [P < 0.05 compared to basal (vehicle treated)]. Data represent the mean ± SE of three independent experiments. (B) Top panel: Representative Western blot of nuclear extracts from NHBE cells treated with vehicle or 10 nM 1,25(OH)2D3 for 24 h using C/EBPα, VDR and β-actin antibodies. Lower Panel: Graphic representation of densitometric scans of Western blots. VDR and C/EBPα are significantly induced in NHBE cells by 1,25(OH)2D3 [P < 0.05 compared to basal (vehicle treated)]. Data represent the mean ± SE of three independent experiments. (C) Analysis of mRNA from NHBE cells for C/EBPα and VDR. Top panel: representative RT-PCR. RT-PCR was performed using RNA from NHBE cells that were treated with vehicle (basal) or 10 nM 1,25(OH)2D3 for 24 h. Lower Panel: Quantitation of C/EBPα and VDR mRNA expression. C/EBPα and VDR mRNAs are significantly induced by 1,25(OH)2D3 in NHBE cells (P < 0.05 compared to basal). Results represent the mean ± SE of three independent experiments.

Identification of a C/EBP binding site in the CAMP promoter

Sequence analysis of the CAMP promoter revealed a putative C/EBP site at − 627/−619, adjacent to the VDRE (−615/−600) (Fig. 3A). Mutation of this site within the −693/+17 CAMP promoter construct markedly attenuated the transcriptional response to C/EBPα (Fig. 3B). Mutation of this site also attenuated the response to 1,25(OH)2D3 (Fig. 3B), further indicating functional cooperation between C/EBPα and VDR in the regulation of hCAMP. EMSAs were performed using synthetic oligonucleotides corresponding to the C/EBP binding sequence adjacent to the VDRE and nuclear extracts from C/EBPα transfected cells. An interaction of C/EBPα protein with this C/EBP site in the CAMP promoter was observed (Fig. 3C). Pre-incubation with C/EBPα antibody or cold oligonucleotide depleted the binding of C/EBPα to the labeled probe, indicating the specificity of the interaction (Fig. 3C).

Fig. 3.

Fig. 3.

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Identification of the C/EBP activation domain in the CAMP promoter (A) Schematic of luciferase constructs of the wild type (WT) CAMP promoter and mutation of the C/EBP site (MT). (B) Luciferase assay using A549 cells transfected using WT CAMP promoter or MT CAMP promoter shown in A. Cells were transfected with VDR expression vector and either co-transfected with C/EBPα expression plasmid or vector alone (basal). Transfected cells were treated with vehicle or 10 nM 1,25(OH)2D3 for 24 h. Luciferase activity is represented as fold induction over the control (mean ± SE; three to six observations per group). pRL- TK-Renilla luciferase was co-transfected as an internal control. *P < 0.05 compared with WT 1,25(OH)2D3 treated. + P < 0.05 compared with WT C/EBPα transfected. (C) Identification of C/EBPα binding motif in the CAMP promoter by EMSA. WT oligonucleotide probe labeled with 32P [free Probe (FP); lane 1] was incubated with 5 μg of nuclear protein from A549 cells transfected with the C/EBPα expression vector (lane 2). The wild-type probe was incubated with C/EBPα antibody in the presence of 5 μg of nuclear protein (lane 3 and 4). The wild-type probe in presence of nuclear protein was also incubated with a 100-fold molar excess of wild-type (WT) cold competitor oligonucleotide (lane 5 and 6). Nonspecific control (NS; using an irrelevant oligonucleotide probe) is shown in lane 7. 100-fold molar excess of mutated (MT) cold competitor oligonucleotide was incubated with the WT probe and 5 μg nuclear protein (lane 8). Gel mobility shift data are representative of at least three experiments.

As the SWI/SNF complex, which facilitates gene transcription by remodeling chromatin using the energy of ATP hydrolysis, has previously been shown to functionally cooperate with C/EBPα in the regulation of gene expression (Inayoshi et al., 2006; Dhawan et al., 2009), we examined a possible role of SWI/SNF in C/EBPα and VDR induced CAMP transcription. The SWI/SNF complex contains one of two homologous ATPases, Brm and the Brahma-related gene 1 (BRG1) (Euskirchen et al., 2012). Previous studies have shown that C/EBPα can interact with both ATPases (Inayoshi et al., 2006). In the presence of Brm-DN (ATPase site mutated Brm, which functions as a DN) there is a dose dependent inhibition of the stimulatory effect of C/EBPα or 1,25(OH)2D3, suggesting the functional involvement of Brm (Fig. 4). ChIP assays were performed using primers designed to amplify the C/EBP/VDR binding site (−627/−600) and nuclear extracts prepared from A549 cells (Fig. 5A) or NHBE cells (Fig. 5B) treated with vehicle or 10 nM 1,25(OH)2D3 (24 h). ChIP assay results indicate enhanced recruitment of C/EBPα and VDR to the CAMP promoter in the presence of 1,25(OH)2D3(Figs.5A and B). ChIP/re-ChIP analysis shows that C/EBPα and Brm bind simultaneously to the CAMP promoter (Figs. 5A and B), suggesting that Brm functionally cooperates with C/EBPα by protein-protein interaction at the C/EBP binding site to regulate CAMP transcription.

Fig. 4.

Fig. 4.

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Functional cooperation between VDR, C/EBPα, and SWI/SNF complex. A549 cells were transfected with the CAMP promoter luciferase construct −693/+17 and VDR (50 ng) or C/EBPα expression plasmid (0.25 μg) in the presence or absence of increasing concentrations of dominant negative (DN) Brahma (Brm) (0.05 μg to 0.25 μg). Brm-DN resulted in a significant decrease in the induction of CAMP transcription by 1,25(OH)2D3 or C/EBPα at all concentration of Brm-DN used (*P < 0.05 compared to cells treated with 1,25(OH)2D3 or transfected with C/EBPα alone).

Fig. 5.

Fig. 5.

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ChIP analysis indicates that VDR, C/EBPα, and Brm are recruited to the CAMP promoter by 1,25(OH)2D3 in lung epithelial cells. (A) ChIP and ChIP re-ChIP assay in A549 cells. A549 cells were treated with vehicle (control) or 1,25(OH)2D3 (10 nM) for 24 h. Cells were cross-linked and cell lysates were subjected to immunoprecipitation with VDR antibody and C/EBPα antibody. DNA was isolated and PCR (using specific primers designed against the C/EBP/VDR site) was carried out in the linear range of DNA amplification. IgG was used as a control. Right panel: Graphic representation of the ChIP assay results. Lower panel: A ChIP re-ChIP assay was also performed using immunoprecipitation first with C/EBPα antibody (ChIP) and then with Brm antibody (re-ChIP). Data represent the mean ± SE of three independent experiments. (B) ChIP assay and ChIP re-ChIP assay were also performed in NHBE cells as described for A549 cells in 5A.

Discussion

1,25(OH)2D3 has been established as a modulator of the body’s adaptive as well as innate immune systems. We earlier reported that 1,25(OH)2D3 induces the expression of CAMP in bronchial epithelial cells and enhances antimicrobial activity against airway pathogens (Yim et al., 2007). In the present study we explored mechanisms by which 1,25(OH)2D3 mediates regulation of innate immune responses to respiratory infections. We report for the first time that transcription factor C/EBPα is induced by 1,25(OH)2D3 in lung epithelial cells and is a potent enhancer of CAMP transcription. Our findings indicate that C/EBPα induces CAMP transcription as well as augments the 1,25(OH)2D3 induction of CAMP transcription in lung epithelial cells. In addition, Brm, a component of the SWI/SNF complex, was found to cooperate with C/EBPα and VDR in the regulation of CAMP. Thus, our findings identify C/EBPα as a 1,25(OH)2D3 target in lung epithelial cells and indicate that C/EBPα, the SWI/SNF complex and 1,25(OH)2D3 may be key factors involved in the regulation of CAMP and therefore in the regulation of specific defense mechanisms in response to respiratory infection.

CAMP expression is known to be strongly stimulated by 1,25 (OH)2D3 in several cell types (White, 2010). In monocytes activation of the pathogen recognition receptor toll-like receptor 2/1 (TLR2/1) in combination with 1,25(OH)2D3 stimulates the expression of CAMP and promotes monocyte killing of Mycobacterium tuberculosis (Liu et al., 2006). In keratinocytes 1,25(OH)2D3 induction of CAMP correlates with increased antimicrobial activity against Staphylococcus aureus (Schauber et al., 2006). 1,25(OH)2D3 increases CD14 and TLR2/1 expression in vitro and in vivo in keratinocytes resulting in enhancement of keratinocyte antimicrobial defense (Schauber et al., 2008). In trophoblasts 1,25(OH)2D3 produced in situ increases the expression of CAMP independent of TLR signaling pathways, suggesting a role for 1,25(OH)2D3 as an autocrine/paracrine regulator in immunity during pregnancy (Liu et al., 2009). The induction of CAMP by 1,25(OH)2D3 in human lung epithelial cells, which correlates with increased antibacterial activity and, unlike keratinocytes and monocytes, is independent of the TLR2/1 signaling pathway (Yim et al., 2007; Hansdottir et al., 2008), suggests a therapeutic benefit of vitamin D in treating airway infections and disorders. Although collectively these findings provide evidence that a key nonclassical function of 1,25(OH)2D3, is to enhance innate immunity through induction of CAMP, the underlying mechanisms of regulation of CAMP have remained poorly understood. A consensus VDRE had previously been identified in the CAMP promoter (at −615 from the TSS) (Larrick et al., 1995; Wang et al., 2004; Gombart et al., 2005; Weber et al., 2005). This VDRE is imbedded in an Alu repeat (a human/primate specific transposable element) (Gombart et al., 2009), suggesting an enhanced role for vitamin D in the regulation of innate immunity in humans and primates. Although CAMP mRNA and protein expression is strongly stimulated by physiological concentrations of 1,25(OH)2D3 in several cell types (Wang et al., 2004; Yim et al., 2007; Schauber et al., 2008), studies by us and others have noted, using promoter-reporter constructs, that the maximum induction of CAMP transcription by 1,25(OH)2D3 (at 10 nM [physiological concentration; Fig. 1B] in lung epithelial cells or at 100 nM in other cell types [VDR transfected COS-7 cells or U937 cells]) (Wang et al., 2004; Gombart et al., 2005; Weber et al., 2005) is only 2–3 fold. The minimal transcriptional responsiveness to 1,25(OH)2D3 compared to endogenous changes in mRNA and/or protein may be due to a more complex regulation of CAMP gene activity by 1,25(OH)2D3. Additional upstream or downstream vitamin D responsive elements not included in our constructs may be involved in 1,25(OH)2D3 regulation of CAMP transcription. It is also possible that the regulation of CAMP by 1,25(OH)2D3 may be post transcriptional as well as transcriptional. Complex cell type specific regulation of CAMP has previously been observed. In keratinocytes for example butyrate alone does not change CAMP mRNA abundance. However butyrate can amplify 1,25(OH)2D3 induction of CAMP mRNA expression (Schauber et al., 2008). In HT-29 colonic epithelial cells the opposite effect was reported. CAMP promoter activity and gene expression are induced by butyrate, although 1,25(OH)2D3 has no effect (Termen et al., 2008). However, in the presence of butyrate 1,25(OH)2D3 significantly augments CAMP promoter activity and this augmentation is dependent on the presence of the VDRE in the promoter construct (Termen et al., 2008). Thus, multiple cell type specific mechanisms, which need to be further defined, are involved in 1,25(OH)2D3 mediated induction of CAMP.

In this study we found that C/EBPα is induced by 1,25 (OH)2D3 in lung epithelial cells, is a potent enhancer of CAMP transcription and augments the 1,25(OH)2D3 induction of CAMP transcription. The C/EBP family of transcription factors has been found to play a key role in mediating the regulation of numerous cellular processes including differentiation, immune and inflammatory processes and hormonal control of metabolism (Poli, 1998; Ramji and Foka, 2002). Different C/EBP isoforms have been reported to show cell type and gene specific regulation of transcription and function (Ramji and Foka, 2002). There is increasing evidence that specific C/EBP family members may be key mediators of 1,25(OH)2D3 action in different cells. We earlier reported that C/EBPβ and not C/EBPα is induced by 1,25(OH)2D3 in kidney and osteoblastic cells and cooperates with 1,25(OH)2D3 to enhance the transcription of CYP24A1, the enzyme involved in the catabolism of 1,25(OH)2D3 (Dhawan et al., 2005). We found that C/EBPβ is unable to enhance CYP24A1 transcription in the absence of 1,25(OH)2D3 and VDR, suggesting, unlike the regulation of the CAMP gene by C/EBPα, that ligand bound VDR/RXR is required to recruit obligate C/EBPβ interacting proteins or transcription factors needed for C/EBPβ enhancement of CYP24A1 transcription (Dhawan et al., 2005). In MCF-7 breast cancer cells we found that C/EBPα but not C/EBPβ is induced by 1,25(OH)2D3 and is a potent enhancer, in the absence of 1,25(OH)2D3, of VDR transcription (Dhawan et al., 2009). In lung epithelium C/EBPα, β and δ are expressed (Cassel and Nord, 2003). C/EBPα plays a crucial role in maturation of the respiratory epithelium as mice with deletion of C/EBPα specifically in respiratory epithelial cells die at birth from respiratory failure (Martis et al., 2006; Roos et al., 2012). In addition, C/EBPα has an important role in the regulation of pulmonary gene expression. The synthesis of surfactant proteins including SP-A, SP-B, SP-C, and SP-D has been reported to be dependent on the expression of C/EBPα (Martis et al., 2006). With regard to regulation of CAMP, C/EBPε, a myeloid specific transcription factor expressed primarily during granulocytic differentiation, activates CAMP expression in myeloid cells (Gombart et al., 2001). C/EBPα is involved in the induction of CAMP mRNA and protein expression in response to endoplasmic reticulum (ER) stress in keratinocytes (Park et al., 2011). ER stress was found to suppress 1,25 (OH)2D3 activation of a VDRE responsive luciferase construct, suggesting that ER stress induction of CAMP expression, mediated by C/EBPα, is independent of VDR in keratinocytes (Park et al., 2011). We report that in lung epithelial cells C/EBPα is a potent enhancer of CAMP transcription, is induced by 1,25(OH)2D3 and cooperates with 1,25(OH)2D3 to induce CAMP transcription. We identified a C/EBP site at −627/−619, which has homology to the C/EBP site in the 25-hydroxyvitamin D3 1α(OH) ase promoter. (Esteban et al., 2004; Stoffels et al., 2006; Zhong et al., 2009), and is adjacent to the CAMP VDRE (at −615/−600). Mutation of this site attenuated the transcriptional response to 1,25(OH)2D3 as well as to C/EBPα, further indicating (unlike the response to ER stress in keratinocytes) cooperation between C/EBPα and VDR in the regulation of CAMP in lung epithelial cells. These findings are consistent with previous studies which showed that when the SmaI site, which is located immediately adjacent and 5’ to the VDRE, is used to generate a deletion mutant [pXP2-CAMP (ΔSmaI)] the construct, which possesses the VDRE, but lacks the C/EBP site, is not activated by 1,25 (OH)2D3 (Gombart et al., 2005). Thus, our study indicates a role for C/EBPα in the regulation of CAMP gene expression in lung epithelial cells and that C/EBPα as well as CAMP are 1,25 (OH)2D3 targets in these cells. In addition these findings, together with our previous findings, provide evidence of a fundamental role for cooperative effects and cross talk between the C/EBP family of transcription factors and VDR in the regulation by 1,25(OH)2D3 of multiple target genes with diverse functions in different cell types.

We also demonstrate in this study that DN Brm inhibits C/EBPα and 1,25(OH)2D3 induction of CAMP promoter transcription and that, in the presence of 1,25(OH)2D3, C/EBPα, and Brm bind simultaneously to the C/EBP binding site in CAMP promoter. These findings suggest that Brm functionally cooperates with C/EBPα and VDR to regulate CAMP transcription. Functional interactions between C/EBPα and SWI/SNF family members have previously been reported for the regulation of other genes in different cell types. We previously reported that Brm cooperates with C/EBPα to modulate the transcription of the hVDR gene in MCF-7 breast cancer cells (Dhawan et al., 2009). C/EBPα has also been reported to functionally recruit Brm during the adipocyte differentiation to regulate the expression of adipocyte specific genes (Pedersen et al., 2001). Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear hormone receptor that regulates adipogenesis. SWI/SNF enzymes have been shown to cooperate with C/EBPα in the regulation of PPARγ transcription (Salma et al., 2004). Although the exact mechanisms involved in cooperation between C/EBPα and Brm in the regulation of CAMP have not as yet been identified, it is possible, similar to the regulation of other genes (Inayoshi et al., 2006), that C/EBPα, by interacting with Brm, induces a conformational change resulting in the stimulation of the ATPase thus facilitating chromatin remodeling and enhanced CAMP transcription.

These mechanisms of enhanced activation of the CAMP gene in lung epithelial cells involving C/EBPα, SWI/SNF, and VDR provide a new understanding of the regulation of innate immunity in the airway and suggest potential candidates for adjunct therapy to prevent and treat airway infection.

Acknowledgments

This work was supported by NIH grants AI-100379 (to S.C. and G. D.), DK-38961 (to S.C.), and AI-065604 (to A.F.G.).

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