C/EBPa and the Vitamin DReceptor Cooperate in theRegulation of Cathelicidin inLung Epithelial CellsPUNEET DHAWAN,1 RAN WEI,1 CHENG SUN,1 ADRIAN F. GOMBART,2

H. PHILLIP KOEFFLER,3 GILL DIAMOND,4 AND SYLVIA CHRISTAKOS1*1Department of Biochemistry and Molecular Biology, Rutgers, the State University of New Jersey, New Jersey Medical School,

Newark, New Jersey2Linus Pauling Institute, Department of Biochemistry and Biophysics, Oregon State University Corvallis, Oregon3Division of Hematology/Oncology, Cedars-Sinai Medical Center, UCLA School of Medicine, Los Angeles, California4Department of Oral Biology, University of Florida, College of Dentistry, Gainsville, Florida

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) and the vitamin D receptor (VDR) have been reported to have an important role in theregulation of innate immunity.We earlier reported that the antimicrobial peptide cathelicidin is induced by 1,25(OH)2D3 in normal humanbronchial epithelial cells with a resultant increase in antimicrobial activity against airway pathogens. In this study, we demonstrate thatC/EBP alpha (C/EBPa) is a potent enhancer of human cathelicidin antimicrobial peptide (CAMP) gene transcription in human lung epithelialcells. In addition we found that C/EBPa functionally cooperates with VDR in the regulation of CAMP transcription. A C/EBP binding sitewas identified at �627/�619 within the CAMP promoter, adjacent to the vitamin D response element (VDRE; �615/�600). Mutation ofthis site markedly attenuated the transcriptional response to C/EBPa as well as to 1,25(OH)2D3, further indicating cooperation betweenthese two factors in the regulation of CAMP. ChIP analysis using 1,25(OH)2D3 treated human lung epithelial cells showedC/EBPa and VDRbinding to the CAMP promoter. C/EBPa has previously been reported to cooperate with Brahma (Brm), an ATPase that is component ofthe SWI/SNF chromatin remodeling complex.We found that dominant negative Brm significantly inhibited C/EBPa as well as 1,25(OH)2D3mediated induction of CAMP transcription, suggesting the functional involvement of Brm. These findings define novel mechanisms involvingC/EBPa, SWI/SNF, and 1,25(OH)2D3 in the regulation of CAMP in lung epithelial cells. These mechanisms of enhanced activation of theCAMP gene in lung epithelial cells suggest potential candidates for the development of modulators of innate immune responses for adjuncttherapy in the treatment of airway infections.

J. Cell. Physiol. 230: 464–472, 2015. © 2014 Wiley Periodicals, Inc.

The respiratory epithelium is exposed to a large number ofpotentially pathogenic microorganisms. A principal defensemechanism protecting the lungs against infection is theproduction of antimicrobial peptides including cathelicidins(Singh et al., 2000; Laube et al., 2006). Since bacteria do notdevelop resistance to antimicrobial peptides, there is increasedinterest in these antimicrobial agents due to their potential astherapy against antibiotic resistant pathogens (Ahmad et al.,2012). Cathelicidins are a family of proteins that originate froma precursormolecule that contains a conservedN-terminal anda less conserved C-terminal cationic antimicrobial peptidedomain 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 humancathelicidin antimicrobial peptide (CAMP) gene. CAMP wasfirst identified in specific granules of neutrophils and wassubsequently identified in monocytes and macrophages,dendritic cells, lymphocytes, mesenchymal stem cells, bonemarrow stroma and epithelial cells of the skin, gastrointestinaltract and respiratory tract. CAMP has broad spectrum activityagainst Gram positive and Gram negative microorganisms(Vandamme et al., 2012). CAMP has also been to found to act asa chemokine, to modulate dendritic cell maturation, topromote 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 resistantpathogens, one approach to fight bacterial infections is toinduce endogenous expression of antimicrobial peptides fortherapeutic benefit (Ahmad et al., 2012). Although 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) was shown to be a potentinducer of CAMP in myeloid cells and in keratinocytes (Wanget al., 2004; Gombart et al., 2005; Weber et al., 2005), theeffects of 1,25(OH)2D3 in airway epithelial cells had notbeen examined. We previously demonstrated that CAMPmRNA and hCAP18 are induced by 1,25(OH)2D3 in humanbronchial epithelial cells and that airway surface fluid from 1,25(OH)2D3 treated cells exhibits increased antimicrobial activity,

Contract grant sponsor: NIH;Contract grant numbers: AI-100379, DK-38961, AI-065604.

*Correspondence to: Dr. Sylvia Christakos, Rutgers, the StateUniversity of New Jersey, New Jersey Medical School, Departmentof Biochemistry and Molecular Biology, 185 South Orange Ave.Newark, NJ 07103. E-mail: christak@njms.rutgers.edu

Manuscript Received: 19 May 2014Manuscript Accepted: 25 July 2014

Accepted manuscript online in Wiley Online Library(wileyonlinelibrary.com): 30 July 2014.DOI: 10.1002/jcp.24729

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indicating CAMP up-regulation correlated with antibacterialactivity induction (Yim et al., 2007). Vitamin D is known to havean important role in calcium homeostasis and bonedevelopment and maintenance. However, recent evidenceprovides evidence for an interrelationship between vitamin Dand health beyond bone, including effects on immune function(Christakos and DeLuca, 2011). Epidemiological studies haveindicated that vitamin D deficiency predisposes humans torespiratory tract infection and that vitamin D sufficiency isassociated with increased resistance to these infections(Hansdottir and Monick, 2011). However the mechanisms arenot understood. The actions of 1,25(OH)2D3 are mediated,similar to other steroids, by the nuclear receptor (VDR) whichheterodimerizes with the retinoid x receptor and interactswith DNA sequences (vitamin D response elements) in targetgenes (Christakos, 2008; Pike and Meyer, 2010). It has beensuggested that cell and promoter specific functions of VDR aremediated through differential recruitment of coactivators andthat increased interaction between VDR and coactivators maybe a major mechanism the couples extracellular signals tovitamin D action (Christakos, 2008; Pike and Meyer, 2010).Understanding tissue specific factors that regulate CAMP geneexpression may be the key to therapeutic manipulation ofendogenous CAMP expression.

In this study we provide evidence that C/EBPa, which isinduced by 1,25(OH)2D3 in lung epithelial cells, is a potentenhancer of CAMP transcription and that C/EBPa cooperateswith VDR and Brahma (Brm) (an ATPase that is a componentof the SWI/SNF chromatin remodeling complex) in theregulation of CAMP transcription. Our findings define novelmechanisms involving C/EBPa, the SWI/SNF complex and1,25(OH)2D3 in the regulation of CAMP gene expression inlung epithelial cells and suggest candidates for thedevelopment of modulators of innate immune responses toairway infection.

Experimental ProceduresMaterials

[a-32P]ATP (3,000 Ci [111 TBq]/mmol) was purchased fromNEN Life Science Products (Boston, MA). The polyvinylidenedifluoride (PVDF) membranes and the pre-stained proteinmolecular weight markers were purchased from Bio-RadLaboratories, Inc. (Hercules, CA). Primary antibodies againstC/EBPa VDR, Brm, and b-actin, and secondary antibodiesagainst mouse and rabbit antisera were obtained from SantaCruz Biotechnology (Santa Cruz, CA). 1,25(OH)2D3 waspurchased from Cayman Chemicals (Ann Arbor, MI).

Cell culture

Cell culture media DMEM and 0.25% trypsin-EDTA andpenicillin-streptomycin-neomycin (PSN) antibiotic mixturewere purchased from Life Technologies (Carlsbad, CA). A549adenocarcinomic human alveolar basal epithelial cells andBEAS-2B human bronchial epithelial cells were obtained fromAmerican Type Culture Collection (Manassas, VA). Normalhuman bronchial epithelial (NHBE) cells were obtained fromLonza, Inc. (Walkersville, MD). Fetal bovine serum (FBS) andcharcoal-stripped FBS were from Gemini Biological Products(Calabasas, CA). A549 cells and BEAS-2B cells were cultured inDMEM supplemented with 10% heat-inactivated FBS and 1%antibiotic mixture PSN. NHBE cells were cultured in BEGM(Bronchial Epithelial Cell Growth Medium) supplemented withGrowth Factors (Lonza, Inc., Walkersville, MD). Cells weregrown in a humidified incubator with atmosphere of 95% air-5% CO2 at 37 °C. For treatments, cells were grown to desiredconfluence and their medium was changed to mediumsupplemented with 2% charcoal-dextran-treated FBS.

Treatments with vehicle or 1,25(OH)2D3 were done for thedurations and with concentrations described in the figurelegends.

Plasmids, transfections and assay ofluciferase activity

The luciferase reporter construct of human CAMP promoter(–693 to þ17) was generated as previously described(Gombart et al., 2005). The C/EBP site at �627/�619 wasmutated by site directed mutagenesis using site directedmutagenesis kit from Stratagene. The oligonucleotides used togenerate the mutated C/EBPa site (C/EBP site shown in bold,mutated nucleotides underlined) were as follows: 5'-AACTGCAACTTACTCTTCCCGGGTTCAATGG-3' and5'-CCATTGAACCCGGGAAGAGTAAGTTGCAGTT-3'.The wild type and the mutant promoter constructs were usedfor reporter assays in A549 human alveolar basal epithelial cellsand BEAS-2B human bronchial epithelial cells. Due to lowtransfection efficiency, NHBE cells were not used for studiesexamining transcriptional regulation of CAMP usingtransfected cells. The C/EBPa, b, and d expression vectorswere a gift of SimonWilliams, Texas Tech University (Lubbock,TX). The dominant negative (DN) C/EBP construct was a giftfrom Charles Vinson (National Cancer Institute, Bethesda,MD). pCMV-Brm and pCMV-mutant Brm (with the ATPase sitemutated that acts as dominant negative inhibitor) wereobtained 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 VDRwere previously reported in A549 cells (Kim et al., 2012), VDRwas cotransfected in studies examining the effect of 1,25(OH)2D3 on CAMP transcription. Empty vectors weretransfected to keep the total DNA concentration equal. Cellswere transfected using Lipofectamine 2000 (Invitrogen)treated as described in Results in the appropriate mediumsupplemented with 2% charcoal-dextran-treated FBS. Aftertreatment with vehicle or the compounds noted at theconcentrations and times indicated in Results, cells wereharvested and a dual luciferase assay was performed accordingto 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 mutantC/EBP site (forward 5'-AACTGCAACTTACTCTTCC-CGGGTTCAATGG-3' and reverse 5'-CCATTGAACCCGG-GAAGAGTAAGTTGCAGTT-3') were synthesized by theRutgers-New Jersey Medical School Molecular ResourceFacility, Newark, New Jersey. The complementary oligos wereannealed, 5' end-labeled with 32P-ATP, purified and used forelectrophoretic mobility shift assay (EMSA) as describedpreviously (Dhawan et al., 2005). Briefly, 5mg of the nuclearextracts from C/EBPa-transfected cells were incubated for20min at 25 °C with 2mg of poly (dI/dC) with or withoutunlabeled specific or nonspecific DNA competitor or C/EBPaantibody in binding buffer (4mM Tris-HCl [pH 7.9], 1mMEDTA [pH 8.0], 60mM KCl, 12% glycerol, 12mM HEPES, and1mM dithiothreitol). This was further incubated with 0.5 ng ofthe labeled oligonucleotide probe (approx. 100,000 cpm) for30min at 25 °C. The samples were electrophoresed for 2.5 hat 4 °C on a 6% nondenaturing polyacrylamide gel that hadbeen pre-electrophoresed for 30min at 100 V/cm at 4 °C in45mM Tris-45mM boric acid-1mM EDTA. The gel wasdried and exposed to X-ray film at –80 °C with intensifyingscreens.

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Nuclear extracts

Nuclear extracts were prepared using the NE-PER nuclear andcytoplasmicextraction reagent kit (ThermoScientific, Rockford,IL) as per manufacturer's protocol. Protease inhibitor cocktail“Complete Mini” was also used during the process (RocheDiagnostics, Indianapolis, IN). The protein concentration of thenuclear 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 isolatetotal RNA from cultured cells. For semi quantitative RT-PCRanalysis of C/EBPa and VDR mRNA levels, RT-PCR wasperformed using 4mg total RNA and Superscript III Reversetranscriptase with Ampli Taq gold DNA polymerase (LifeTechnologies). Primers used were as follows: C/EBPa forward5'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); b-actin forward5'-CCTGTGGATCTGACAGCTGAA-3' (35 cycles) and re-verse 5'-TCCCAAATCGGTTGGAGATA-3' (35 cycles);GAPDH forward 5'-TCACCATCTTCCAGGAGCG-3' andreverse 5'-CTGCTTCACCACCTTCTTGA-3' (35 cycles). Thecycles were chosen so that the amplification was conducted inthe linear range of amplification efficiency. The resulting PCRproducts were subjected to electrophoresis on a 1% agarose gelcontaining ethidium bromide and bands were visualized underUV light. Gel data were recorded using the Gene GeniusbioImaging System (Syngene, Frederick MD) and relativedensities of the bands were determined using ImageJ software(NIH, Bethesda, MD). Data were normalized for the expressionof b-actin mRNAwithin the sample. Real time quantitative PCR(RTQ-PCR) was also used to quantify CAMP mRNA in NHBEcells usingMyCycler (Bio-Rad Laboratories, Hercules, CA) using2� SYBR Green PCR Master Mix (Applied Biosystems, FosterCity, CA) in a volume of 20ml. RTQ-PCR primers used were:hCAMPforward,5'-GTCACCAGAGGATTGTGACTTCAA-3'and reverse, 5'-TTGAGGGTCACTGTCCCCATA-3'; b actinforward, 5'-AGTCTGTGGCATCCACGAAACTAC-3', andreverse, 5'-CTTCTGCATCCTGTCGGCAATG-3'. The PCRfragmentswere amplified for 45 cycles (15 sec at 95 °Cand 1minat 60 °Cwhich providedoptimalmelt curves for all primer pairs).The relative CAMP mRNA expression in response to 1,25(OH)2D3was calculated using the 2

-DDCtmethod, normalized tob-actin and presented as fold induction.

Chromatin immunoprecipitation (ChIP) assay

A549 cells or NHBE cells, treated with vehicle or 1,25(OH)2D3were used for the Chromatin immunoprecipitation (ChIP)assays as previously described (Shen and Christakos, 2005;Dhawan and Christakos, 2010). In brief, treated cells werewashed with PBS and cross-linked using 1% formaldehyde for15min. Crosslinking was stopped by adding glycine to a finalconcentration of 0.125M. The cells were washed twice withice-cold PBS and were collected by scraping. The cells werelysed for 20min each, first in buffer 1 (5mM Pipes, pH 8.0,85mM KCl, 0.5% Nonidet P-40) followed by buffer 2 (1% SDS,10mM EDTA, 50mM Tris-HCl, pH 8.1). The resultingchromatin pellet was sonicated to an average DNA size of500 bp (evaluated by 1% agarose gel electrophoresis) using aFisher model 100 sonic dismembranator at a power setting oftwo. The sonicated extract was centrifuged for 10min at13,000 rpm at 4 °C and then diluted into ChIP dilution buffer

(16.7mM Tris-HCl, pH 8.1, 150mM NaCl, 0.01% SDS, 1.1%Triton X-100, and 1.2mM EDTA). Immunoprecipitations wereperformed at 4 °C overnight with the indicated antibody. After4 h incubation with protein A agarose (RocklandImmunochemicals, Inc., Gilbertsville, PA), the beads werecollected by centrifugation. The protein A agarose beads werewashed sequentially as previously described (Shen andChristakos, 2005; Dhawan and Christakos, 2010). Theprotein-DNA was then eluted by using 1% SDS and 0.1MNaHCO3 for 15min twice. Cross-links were reversed byincubating at 65 °C overnight in elution buffer with 0.2MNaCl.DNA fragments were purified using the Qiagen QIAquick PCRpurification kit (Valencia, CA) and PCR was performed usingprimers designed to amplify fragments of human CAMPpromoter 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 wasperformed in the linear range of DNA amplification. 250 bpPCR products were resolved in 1% agarose gel and visualizedusing ethidium bromide staining. DNA obtained beforeprecipitation was used as the input. 10% of input was usedfor PCR reaction. DNA acquired from immunoprecipitatesperformed with IgG was subjected to PCR using the primersdesigned to amplify the fragment containing the C/EBP/VDRbinding site to exclude nonspecific binding.

Re-ChIP experiments were also performed using sequentialchromatin immunoprecipitation and two different antibodies(a-C/EBPa and a-Brm) to assay for the simultaneous presenceof these two factors at the same site in the CAMP promoter. InRe-ChIP experiments, on the second day of the ChIPexperiment, complexes were eluted in 60ml of elution buffercontaining 10mM dithiothreitol for 30min at 37 °C. The elutedsamples were diluted 50 times with ChIP dilution buffer andsubjected again to the ChIP procedure using the secondantibody (a-Brm).

Statistical analysis

Results are expressed as means� standard errors (SE), andsignificance was determined by analysis by Student's t-test fortwo-group comparison or by analysis of variance formultiple-group comparison.

ResultsVDR and C/EBPa cooperate in the regulation of CAMPtranscription in lung epithelial cells

Although 1,25(OH)2D3 induces antimicrobial peptides inmyeloid cells and keratinocytes, very little is known about theeffect of 1,25(OH)2D3 on antimicrobial activity and themechanisms by which 1,25(OH)2D3 modulates innateimmunity in airway epithelial cells. We previously reported theinduction of CAMP by 1,25(OH)2D3 in NHBE cells and theresultant increase in antimicrobial activity against airwaypathogens (Yim et al., 2007; Fig. 1A). To examine themechanism of activation of CAMP gene expression by 1,25(OH)2D3, A549 human pulmonary epithelial cells weretransfected with the CAMP promoter (�693/þ 17) luciferaseconstruct and treated with 1,25(OH)2D3. A concentrationdependent 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). Becausesequence analysis indicated putative C/EBP binding sites in theCAMP promoter, we investigated the possibility that C/EBPsmay be involved in the regulation of CAMP transcription.C/EBPa (0.025–0.25mg) significantly enhanced CAMPtranscription (3.6–8.4 fold; P< 0.05 compared to vectortransfected control) (Fig. 1C). Although 1,25(OH)2D3 (10 nM)or C/EBPa (0.1mg) stimulates CAMP transcription 2-, 3- and

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Fig. 1. Functional cooperation between VDR and C/EBPa 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 (10nM for 6 and 24h). *P< 0.05 compared to zerotime. Lower concentrations of 1,25(OH)2D3 (1 nM and below for 6 and 24h) fail to elicit a response; Yim et al. (2007). (B) 1,25(OH)2D3 inducesCAMP transcription in human pulmonary epithelial cells. Cells were transfected with the CAMP promoter (–693/þ 17) (250ng) and VDRexpression plasmid (50ng) and treated with 1,25(OH)2D3 (1 nM to 100nM) for 24h. CAMP promoter activity is represented as fold induction(mean�SE; n¼ 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/þ 17) and increasing concentrations of C/EBPa. *P< 0.05 compared tobasal. (D) C/EBPa induces CAMP transcription and cooperates with 1,25(OH)2D3 in the regulation of CAMP transcription. Top panel: C/EBPapromoter schematic showing the location of the VDRE (�615/�600) and the putative C/EBP binding site (�627/�619). Left panel: A549 cellswere transfected with the CAMP promoter (–693/þ 17) (250ng), VDR (50ng) and C/EBPa (0.1mg) or C/EBPb (0.1mg) 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 (10nM)for 24h. CAMP promoter activity is expressed as fold induction (mean�SE; n¼ 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 isexpressed as fold induction (mean�SE; n¼ 3–4 separate experiments) by comparison to basal. *P< 0.05 compared to basal. The induction ofCAMP promoter activity by the combination of 1,25(OH)2D3 and C/EBPa is significantly greater than CAMP promoter activity in response toC/EBPa alone or 1,25(OH)2D3 alone,þP< 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.

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6-fold, respectively, C/EBPa transfection in A549 cellscombined with 1,25(OH)2D3 treatment resulted in a 13 foldinduction of CAMP transcription, suggesting functionalcooperation between C/EBPa and 1,25(OH)2D3 in theregulation of CAMP transcription (Fig. 1D, left panel). UnlikeC/EBPa, C/EBPb did not significantly affect CAMPtranscription (Fig. 1D, left panel). C/EBPdwas also not found tostimulate CAMP transcription (not shown). Similar resultswere observed using BEAS-2B cells (Fig. 1D, right panel). Inaddition, RT-PCR analysis indicated that CAMP geneexpression was induced by 1,25(OH)2D3, C/EBPa and thecombination of C/EBPa and 1,25(OH)2D3 in A549 cells (Fig. 1E,left panel) and in BEAS-2B cells (Fig. 1E, right panel), consistentwith a role of C/EBPa alone and in cooperationwith VDR in theregulation of CAMP gene expression.

C/EBPa expression is induced by 1,25(OH)2D3 in humanlung epithelial cells

Since we previously reported the regulation of C/EBP familymemberby1,25(OH)2D3 indifferent cell types,weexamined thepossibility that C/EBPa is a 1,25(OH)2D3 target in lung epithelialcells. When A549 cells were treated with increasingconcentrations of 1,25(OH)2D3 Western blot analysis usingnuclear extracts from vehicle (Basal) or 1,25(OH)2D3 treatedcells showed a significant induction of C/EBPa expression at 1,10, and 100nM1,25(OH)2D3 (Fig. 2A). The induction ofC/EBPainA549cellswasaccompaniedbyan increase in theexpressionof

VDR (Fig. 2A). C/EBPa and VDR were also significantly inducedby 1,25(OH)2D3 (10nM 1,25(OH)2D3 treatment for 24 h) inNHBE cells (Fig. 2B). Induction of C/EBPa and VDR mRNA by1,25(OH)2D3 in NHBE cells is shown in Fig. 2C.

Identification of a C/EBP binding site in the CAMPpromoter

Sequence analysis of the CAMP promoter revealed a putativeC/EBP site at – 627/�619, adjacent to the VDRE (�615/�600)(Fig. 3A). Mutation of this site within the �693/þ 17 CAMPpromoter construct markedly attenuated the transcriptionalresponse to C/EBPa (Fig. 3B). Mutation of this site alsoattenuated the response to 1,25(OH)2D3 (Fig. 3B), furtherindicating functional cooperation between C/EBPa and VDR inthe regulation of hCAMP. EMSAs were performed usingsynthetic oligonucleotides corresponding to the C/EBP bindingsequence adjacent to the VDRE and nuclear extracts fromC/EBPa transfected cells. An interaction of C/EBPa proteinwith this C/EBP site in the CAMP promoter was observed(Fig. 3C). Pre-incubation with C/EBPa antibody or coldoligonucleotide depleted the binding of C/EBPa to the labeledprobe, indicating the specificity of the interaction (Fig. 3C).

As the SWI/SNF complex, which facilitates gene transcriptionby remodeling chromatin using the energy ofATPhydrolysis, haspreviously been shown to functionally cooperatewithC/EBPa inthe regulation of gene expression (Inayoshi et al., 2006; Dhawanet al., 2009), we examined a possible role of SWI/SNF inC/EBPa

Fig. 2. C/EBPa 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 100nM) for 24h and probed with C/EBPa, VDR and b-actin antibodies. Lower Panel: Graphic representation ofdensitometric scans of Western blots. C/EBPa and VDR are significantly induced by 1, 10, and 100nM 1,25(OH)2D3 [P< 0.05 compared tobasal (vehicle treated)]. Data represent the mean�SE of three independent experiments. (B) Top panel: Representative Western blot ofnuclear extracts fromNHBE cells treated with vehicle or 10nM 1,25(OH)2D3 for 24h using C/EBPa, VDR and b-actin antibodies. Lower Panel:Graphic representation of densitometric scans of Western blots. VDR and C/EBPa 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 fromNHBE cells for C/EBPa and VDR. Top panel: representative RT-PCR. RT-PCR was performed using RNA from NHBE cells that were treatedwith vehicle (basal) or 10 nM 1,25(OH)2D3 for 24h. Lower Panel: Quantitation of C/EBPa and VDR mRNA expression. C/EBPa and VDRmRNAs are significantly induced by 1,25(OH)2D3 in NHBE cells (P< 0.05 compared to basal). Results represent the mean�SE of threeindependent experiments.

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and VDR induced CAMP transcription. The SWI/SNF complexcontainsoneof twohomologousATPases, Brmand theBrahma-related gene 1 (BRG1) (Euskirchen et al., 2012). Previous studieshave shown that C/EBPa can interact with both ATPases(Inayoshi et al., 2006). In the presence of Brm-DN (ATPase sitemutatedBrm,which functionsasaDN)there isadosedependentinhibition of the stimulatory effect of C/EBPa or 1,25(OH)2D3,suggesting the functional involvement of Brm(Fig. 4).ChIP assayswere performed using primers designed to amplify the C/EBP/VDR binding site (�627/�600) and nuclear extracts preparedfrom A549 cells (Fig. 5A) or NHBE cells (Fig. 5B) treated withvehicle or 10 nM1,25(OH)2D3 (24 h).ChIP assay results indicateenhanced recruitment of C/EBPa and VDR to the CAMPpromoter in thepresenceof1,25(OH)2D3 (Figs. 5AandB).ChIP/re-ChIPanalysis showsthatC/EBPaandBrmbindsimultaneouslyto the CAMP promoter (Figs. 5A and B), suggesting that Brmfunctionally cooperates with C/EBPa by protein-proteininteraction at the C/EBP binding site to regulate CAMPtranscription.

Discussion

1,25(OH)2D3 has been established as a modulator of the body'sadaptive as well as innate immune systems. We earlier

reported that 1,25(OH)2D3 induces the expression of CAMP inbronchial epithelial cells and enhances antimicrobial activityagainst airway pathogens (Yim et al., 2007). In the present studywe explored mechanisms by which 1,25(OH)2D3 mediatesregulation of innate immune responses to respiratoryinfections.We report for the first time that transcription factorC/EBPa is induced by 1,25(OH)2D3 in lung epithelial cells and isa potent enhancer of CAMP transcription. Our findingsindicate that C/EBPa induces CAMP transcription as well asaugments the 1,25(OH)2D3 induction of CAMP transcription inlung epithelial cells. In addition, Brm, a component of the SWI/SNF complex, was found to cooperate with C/EBPa and VDRin the regulation of CAMP. Thus, our findings identify C/EBPaas a 1,25(OH)2D3 target in lung epithelial cells and indicate thatC/EBPa, the SWI/SNF complex and 1,25(OH)2D3 may be keyfactors involved in the regulation of CAMP and therefore in theregulation of specific defense mechanisms in response torespiratory infection.

CAMP expression is known to be strongly stimulated by 1,25(OH)2D3 in several cell types (White, 2010). In monocytesactivation of the pathogen recognition receptor toll-likereceptor 2/1 (TLR2/1) in combination with 1,25(OH)2D3stimulates the expression of CAMP and promotes monocytekilling of Mycobacterium tuberculosis (Liu et al., 2006). In

Fig. 3. 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 MTCAMP promoter shown in A. Cells were transfected with VDR expression vector and either co-transfected with C/EBPa expression plasmidor vector alone (basal). Transfected cells were treated with vehicle or 10nM 1,25(OH)2D3 for 24h. Luciferase activity is represented as foldinduction over the control (mean�SE; three to six observations per group). pRL- TK-Renilla luciferase was co-transfected as an internalcontrol. *P< 0.05 compared with WT 1,25(OH)2D3 treated.þP< 0.05 compared with WT C/EBPa transfected. (C) Identification of C/EBPabinding motif in the CAMP promoter by EMSA. WT oligonucleotide probe labeled with 32P [free Probe (FP); lane 1] was incubated with 5mgof nuclear protein from A549 cells transfected with the C/EBPa expression vector (lane 2). The wild-type probe was incubated with C/EBPaantibody in the presence of 5mg of nuclear protein (lane 3 and 4). The wild-type probe in presence of nuclear protein was also incubated with a100-fold molar excess of wild-type (WT) cold competitor oligonucleotide (lane 5 and 6). Nonspecific control (NS; using an irrelevantoligonucleotide probe) is shown in lane 7. 100-fold molar excess of mutated (MT) cold competitor oligonucleotide was incubated with the WTprobe and 5mg nuclear protein (lane 8). Gel mobility shift data are representative of at least three experiments.

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keratinocytes 1,25(OH)2D3 induction of CAMP correlateswith increased antimicrobial activity against Staphylococcusaureus (Schauber et al., 2006). 1,25(OH)2D3 increases CD14and TLR2/1 expression in vitro and in vivo in keratinocytesresulting in enhancement of keratinocyte antimicrobial defense(Schauber et al., 2008). In trophoblasts 1,25(OH)2D3 producedin situ increases the expression of CAMP independent of TLRsignaling pathways, suggesting a role for 1,25(OH)2D3 as anautocrine/paracrine regulator in immunity during pregnancy(Liu et al., 2009). The induction of CAMP by 1,25(OH)2D3 inhuman lung epithelial cells, which correlates with increasedantibacterial 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 ofvitamin D in treating airway infections and disorders. Althoughcollectively these findings provide evidence that a keynonclassical function of 1,25(OH)2D3, is to enhance innateimmunity through induction of CAMP, the underlyingmechanisms of regulation of CAMP have remained poorlyunderstood. A consensus VDRE had previously been identifiedin 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 ofinnate immunity in humans and primates. Although CAMPmRNA and protein expression is strongly stimulated byphysiological concentrations of 1,25(OH)2D3 in several celltypes (Wang et al., 2004; Yim et al., 2007; Schauber et al., 2008),studies by us and others have noted, using promoter-reporterconstructs, that the maximum induction of CAMPtranscription by 1,25(OH)2D3 (at 10 nM [physiologicalconcentration; Fig. 1B] in lung epithelial cells or at 100 nM inother cell types [VDR transfected COS-7 cells or U937 cells])

(Wang et al., 2004; Gombart et al., 2005; Weber et al., 2005) isonly 2–3 fold. The minimal transcriptional responsiveness to1,25(OH)2D3 compared to endogenous changes in mRNA and/or protein may be due to a more complex regulation of CAMPgene activity by 1,25(OH)2D3. Additional upstream ordownstream vitamin D responsive elements not included inour constructs may be involved in 1,25(OH)2D3 regulation ofCAMP transcription. It is also possible that the regulation ofCAMP by 1,25(OH)2D3 may be post transcriptional as well astranscriptional. Complex cell type specific regulation of CAMPhas previously been observed. In keratinocytes for examplebutyrate alone does not change CAMP mRNA abundance.However butyrate can amplify 1,25(OH)2D3 induction ofCAMP mRNA expression (Schauber et al., 2008). In HT-29colonic epithelial cells the opposite effect was reported. CAMPpromoter 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)2D3significantly augments CAMP promoter activity and thisaugmentation is dependent on the presence of the VDRE in thepromoter construct (Termen et al., 2008). Thus, multiple celltype specificmechanisms, which need to be further defined, areinvolved in 1,25(OH)2D3 mediated induction of CAMP.

In this study we found that C/EBPa is induced by 1,25(OH)2D3 in lung epithelial cells, is a potent enhancer of CAMPtranscription and augments the 1,25(OH)2D3 induction ofCAMP transcription. The C/EBP family of transcription factorshas been found to play a key role in mediating the regulation ofnumerous cellular processes including differentiation, immuneand inflammatory processes and hormonal control ofmetabolism (Poli, 1998; Ramji and Foka, 2002). Different C/EBPisoforms have been reported to show cell type and genespecific regulation of transcription and function (Ramji andFoka, 2002). There is increasing evidence that specific C/EBPfamily members may be key mediators of 1,25(OH)2D3 actionin different cells. We earlier reported that C/EBPb and notC/EBPa is induced by 1,25(OH)2D3 in kidney and osteoblasticcells and cooperates with 1,25(OH)2D3 to enhance thetranscription of CYP24A1, the enzyme involved in thecatabolism of 1,25(OH)2D3 (Dhawan et al., 2005). We foundthat C/EBPb is unable to enhance CYP24A1 transcription inthe absence of 1,25(OH)2D3 and VDR, suggesting, unlike theregulation of the CAMP gene by C/EBPa, that ligand boundVDR/RXR is required to recruit obligate C/EBPb interactingproteins or transcription factors needed for C/EBPbenhancement of CYP24A1 transcription (Dhawan et al., 2005).In MCF-7 breast cancer cells we found that C/EBPa but notC/EBPb is induced by 1,25(OH)2D3 and is a potent enhancer, inthe absence of 1,25(OH)2D3, of VDR transcription (Dhawanet al., 2009). In lung epithelium C/EBPa, b and d are expressed(Cassel and Nord, 2003). C/EBPa plays a crucial role inmaturation of the respiratory epithelium as mice with deletionof C/EBPa specifically in respiratory epithelial cells die at birthfrom respiratory failure (Martis et al., 2006; Roos et al., 2012).In addition, C/EBPa has an important role in the regulation ofpulmonary gene expression. The synthesis of surfactantproteins including SP-A, SP-B, SP-C, and SP-D has beenreported to be dependent on the expression of C/EBPa (Martiset al., 2006). With regard to regulation of CAMP, C/EBPe, amyeloid specific transcription factor expressed primarilyduring granulocytic differentiation, activates CAMP expressionin myeloid cells (Gombart et al., 2001). C/EBPa is involved inthe induction of CAMP mRNA and protein expression inresponse 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/EBPa, is independent of VDR in keratinocytes(Park et al., 2011). We report that in lung epithelial cells

Fig. 4. Functional cooperation between VDR, C/EBPa, and SWI/SNF complex. A549 cells were transfected with the CAMPpromoter luciferase construct –693/þ17 and VDR (50ng) or C/EBPaexpression plasmid (0.25mg) in the presence or absence ofincreasing concentrations of dominant negative (DN) Brahma(Brm) (0.05mg to 0.25mg). Brm-DN resulted in a significantdecrease in the induction of CAMP transcription by 1,25(OH)2D3 orC/EBPa at all concentration of Brm-DN used (*P< 0.05 comparedto cells treated with 1,25(OH)2D3 or transfected with C/EBPaalone).

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C/EBPa is a potent enhancer of CAMP transcription, is inducedby 1,25(OH)2D3 and cooperates with 1,25(OH)2D3 to induceCAMP transcription.We identified a C/EBP site at�627/�619,which has homology to the C/EBP site in the25-hydroxyvitamin D3 1a(OH) ase promoter. (Esteban et al.,2004; Stoffels et al., 2006; Zhong et al., 2009), and is adjacent tothe CAMP VDRE (at �615/�600). Mutation of this siteattenuated the transcriptional response to 1,25(OH)2D3 aswell as to C/EBPa, further indicating (unlike the response to ERstress in keratinocytes) cooperation between C/EBPa andVDR in the regulation of CAMP in lung epithelial cells. Thesefindings are consistent with previous studies which showedthat when the SmaI site, which is located immediately adjacentand 5' to the VDRE, is used to generate a deletion mutant[pXP2-CAMP (DSmaI)] the construct, which possesses theVDRE, but lacks the C/EBP site, is not activated by 1,25(OH)2D3 (Gombart et al., 2005). Thus, our study indicates arole for C/EBPa in the regulation of CAMP gene expression inlung epithelial cells and that C/EBPa 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 afundamental role for cooperative effects and cross talkbetween the C/EBP family of transcription factors and VDR inthe regulation by 1,25(OH)2D3 of multiple target genes withdiverse functions in different cell types.

We also demonstrate in this study that DN Brm inhibitsC/EBPa and 1,25(OH)2D3 induction of CAMP promoter

transcription and that, in the presence of 1,25(OH)2D3,C/EBPa, and Brm bind simultaneously to the C/EBP binding sitein CAMP promoter. These findings suggest that Brmfunctionally cooperates with C/EBPa and VDR to regulateCAMP transcription. Functional interactions between C/EBPaand SWI/SNF family members have previously been reportedfor the regulation of other genes in different cell types. Wepreviously reported that Brm cooperates with C/EBPa tomodulate the transcription of the hVDR gene in MCF-7 breastcancer cells (Dhawan et al., 2009). C/EBPa has also beenreported to functionally recruit Brm during the adipocytedifferentiation to regulate the expression of adipocyte specificgenes (Pedersen et al., 2001). Peroxisomeproliferator-activated receptor g (PPARg) is a nuclearhormone receptor that regulates adipogenesis. SWI/SNFenzymes have been shown to cooperate with C/EBPa in theregulation of PPARg transcription (Salma et al., 2004).Although the exact mechanisms involved in cooperationbetween C/EBPa and Brm in the regulation of CAMP have notas yet been identified, it is possible, similar to the regulation ofother genes (Inayoshi et al., 2006), that C/EBPa, by interactingwith Brm, induces a conformational change resulting in thestimulation of the ATPase thus facilitating chromatinremodeling and enhanced CAMP transcription.

Thesemechanisms of enhanced activation of theCAMP genein lung epithelial cells involving C/EBPa, SWI/SNF, and VDRprovide a new understanding of the regulation of innate

Fig. 5. ChIP analysis indicates that VDR, C/EBPa, 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 24h. Cells were cross-linked and cell lysates were subjected to immunoprecipitation with VDR antibody and C/EBPa antibody. DNA was isolated and PCR (usingspecific 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 usingimmunoprecipitation first with C/EBPa antibody (ChIP) and then with Brm antibody (re-ChIP). Data represent the mean�SE of threeindependent experiments. (B) ChIP assay and ChIP re-ChIP assay were also performed in NHBE cells as described for A549 cells in 5A.

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immunity in the airway and suggest potential candidates foradjunct therapy to prevent and treat airway infection.

Acknowledgments

This workwas supported byNIH grants AI-100379 (to S.C. andG. D.), DK-38961 (to S.C.), and AI-065604 (to A.F.G.).

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