Physiological Regulation of Uteroglobin/CCSP Expression

181

Physiological Regulation of Uteroglobin/CCSP Expression

ALBERT CHANG,a PATRICIA RAMSAY,b,c BIHONG ZHAO,c MOON PARK,b

SUSAN MAGDALENO,d MICHAEL J. REARDON,a STEPHEN WELTY,b AND FRANCESCO J. DEMAYOb,c,e

aDepartment of Surgery, bDepartment of Pediatrics, cDepartment of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, USAdDepartment of Developmental Neurobiology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA

ABSTRACT: Uteroglobin/CCSP is expressed specifically in the Clara cells. Thisallows the gene to be used as a marker to identify the elements regulating thephysiologic and cell-specific expression of this gene. The regulation of UG/CCSP by IFN-� was shown to be at the level of the proximal promoter by theupregulation of HNF3�. This has allowed the determination of the factorsresponsible for the expression of UG/CCSP.

INTRODUCTION

Uteroglobin was first discovered in the late 1960s as a secretory product of therabbit reproductive tract and lung (for review, see reference 1). This protein is alsoknown as blastokinin,2 uteroglobin,3 urinary protein 1,4 polychlorobiphenyl bindingprotein,5 Clara cell 10-kDa protein (CC10),6 Clara cell 16-kDa protein (CC16),7 andClara cell secretory protein (CCSP).8 For sake of simplicity, this protein will bereferred to as uteroglobin/Clara cell secretory protein (UG/CCSP). Although ex-pressed in a variety of tissues, the major site of expression of this protein in allspecies investigated, with the exception of lagomorphs, is the Clara cells of thelungs.9 The mature form of this protein consists of two identical polypeptides heldtogether in antiparallel orientation by disulfide bridges.10,11 The antiparallel orienta-tion of these peptides forms a hydrophobic pocket that allows this protein to bindcompounds, such as polychlorinated biphenyl compounds,12 progesterone,1 andretinol.13 Among the many biological properties identified with UG/CCSP, severalanti-inflammatory properties have been attributed to this protein. This protein hasbeen shown to inhibit phospholipase A2,14 the production and function of interferongamma (IFN-γ),15 and the neutrophil inflammation and chemotaxic response.5 Ad-ditional evidence that supports this protein playing an important anti-inflammatoryrole lies in its location within the genome. The human UG/CCSP gene is located onchromosome 11q12.3-q13.1.16 In this region, other genes are involved in the regula-

eCorresponding author: Francesco J. DeMayo, Department of Molecular and Cellular Biology,Baylor College of Medicine, 1 Baylor Plaza, M725, Houston, TX 77030. Voice: 713-798-6241;fax: 713-790-1275.

[email protected]

182 ANNALS NEW YORK ACADEMY OF SCIENCES

tion of inflammation. This implies that UG/CCSP plays some role in modulating theinflammatory process.

Although the function of UG/CCSP has not been fully defined, ablation of UG/CCSP by homologous recombination in embryonic stem cells has confirmed the pro-tective properties of UG/CCSP. Two groups, each using different targeting strategiesto disrupt the expression of this gene, have ablated the mouse UG/CCSP gene (mUG/CCSP). Stripp and coworkers inserted the selection marker, PGKneo, into the firstexon of this gene to disrupt expression.17 The phenotype observed in the unchal-lenged mouse was a failure of the lungs to accumulate biphenyl compounds and adisruption of Clara cell morphology at the ultrastructural level. There was no disrup-tion in pulmonary function or animal viability observed by these investigators. How-ever, when the UG/CCSP knockout mice were challenged with a hyperoxic insult,these mice showed significantly reduced survival time when compared to wild-typemice. Surveying the lungs of the UG/CCSP knockout mice after hyperoxic challengedemonstrated elevated levels of the proinflammatory cytokines and the interleukins,IL-6, IL-1b, and IL-3, as compared to wild-type mice simultaneously exposed tohyperoxia. This analysis demonstrated a protective role for mUG/CCSP in responseto hyperoxic insult.18 The protective role may be due to maintenance of Clara cellintegrity provided by UG/CCSP and/or its anti-inflammatory activity. Zhang and co-workers ablated mUG/CCSP expression by deleting part of the second exon andintron.19 Again, these investigators observed no obvious disruption in pulmonaryfunction in the unchallenged mouse. The UG/CCSP knockout mice showed a signif-icant increase in serum phospholipase A activity, supporting the observation thatUG/CCSP is a potent inhibitor of phospholipase A2. However, the major discrepancybetween the model generated by Zhang and coworkers and that by Stripp and co-workers is that the UG/CCSP −/− mice generated by Zhang and coworkers died fromrenal failure secondary to fibronectin accumulation in the glomeruli. The latter phe-notype was confirmed by a transgenic antisense approach.20 Despite the differencein phenotype between the two knockout mouse models, there is no doubt that UG/CCSP provides an important protective role in the lungs. The protective function ofUG/CCSP as well as the cellular specificity in pulmonary epithelium makes UG/CCSP an ideal model to investigate the physiological regulation of CCSP in theClara cells of the lungs. The understanding of the molecular regulation of UG/CCSPwill allow for not only an understanding of UG/CCSP function, but also an under-standing of the biology and differentiation of the pulmonary Clara cell.

CLARA CELLS

First identified as a distinct cell type by Kolliker in 1881, credit was given to MaxClara, who described the same cell type in 1937.21 Clara concluded that the cellswere exocrine secretory cells releasing a substance that was later identified by Kuhnas protein and not mucous in nature.22 The Clara cells are nonciliated, secretory cellslining the bronchioles of the lung.21 The distribution and abundance of Clara cellsthroughout the respiratory tree vary in a species-specific manner.9,23 Clara cells canbe identified in the airways by their distinct dome-shaped morphology and abun-dance of secretory granules.23 Clara cells contain abundant levels of cytochromeP450 mixed-function oxidases, and they secrete surfactant proteins A, B, and D, a

183CHANG et al.: UG/CCSP EXPRESSION

leukocyte protease inhibitor, a trypsin-like protease, and a 16-kDa protein, Clara cellsecretory protein, UG/CCSP.24 Clara cells have an important protective role in air-way physiology. The cytochrome P450 mixed-function oxidases serve to metabolizexenobiotics in the lungs and UG/CCSP serves to protect the lungs against hyperoxicdamage18 and inflammation.19 Due to the cellular preference and high levels of ex-pression of this protein, UG/CCSP has been the subject of numerous investigationsto define its regulation and functional significance. Molecular analysis of UG/CCSPgene expression will define how interactions between the transcription factors regu-lating UG/CCSP gene expression function to coordinate UG/CCSP gene expressionin a cell-specific manner, as well as in response to external stimuli. Investigation ofthe murine gene for UG/CCSP is especially important because the mouse can begenetically manipulated to allow for the investigation of factors that regulate UG/CCSP gene expression.

REGULATION OF UG/CCSP GENE EXPRESSION

Initial investigations of the rabbit UG/CCSP gene showed that steroid hormonesregulated this gene. This steroid hormone regulation of the UG/CCSP gene dependsupon the tissue in which this gene is expressed. In the female reproductive tract, thisgene is regulated by the ovarian steroids, estrogen and progesterone.25–27 In the malereproductive tract, this gene is regulated by androgens.28 In the lungs, UG/CCSPgene expression is influenced by glucocorticoids,25 IFN-γ,29 interleukin-4 (IL-4),30

and hyperoxia.31 Since most species studied express UG/CCSP predominantly in thelung, the investigation of UG/CCSP in the lung is of particular physiological signif-icance. Glucocorticoids have been shown to increase UG/CCSP expression in vivoby administration of dexamethasone to adrenalectomized mice32,33 and in vitro byadministering dexamethasone to transformed mouse lung epithelial cells.34 IFN-γhas been shown to increase UG/CCSP levels both in vivo by intratracheal adminis-tration of IFN-γ to mice and in vitro by the culture of mouse transformed Clara cellsin the presence of IFN-γ.29 Transgenic mice overexpressing IL-4 in the airways havedecreased expression of UG/CCSP30 and mice exposed to chronic hyperoxia havebeen shown to display a significant decrease in UG/CCSP mRNA.31 Interestingly,hyperoxia, which increases the expression of other pulmonary epithelial secretoryproteins (i.e., surfactant proteins A, B, and C), represses UG/CCSP.35 Since UG/CCSP protects the lungs from hyperoxic damage, the repression of UG/CCSP levelsby hyperoxia may worsen the severity of oxygen damage to the lungs. Identificationof the mechanism by which O2 represses UG/CCSP expression may lead to thera-peutic approaches to reduce the severity of hyperoxic damage to the lungs. Molecu-lar analysis of the regulation of the expression of the mouse UG/CCSP gene willallow for an elucidation of these mechanisms.

CHARACTERIZATION OF THE MUG/CCSP GENE

The cloning of the mouse cDNA for the UG/CCSP gene revealed that 288 bp ofsequence coded for the mUG/CCSP protein. Sequence analysis revealed that themUG/CCSP amino acid sequence was 90%, 52%, and 51% homologous to the rat,


human, and rabbit UG/CCSP proteins, respectively.36 The mUG/CCSP cDNA wasexpressed in bacteria and the recombinant protein was used to generate antibodies tothe mUG/CCSP protein. In situ hybridization and immunohistochemical analyseswere used to determine the developmental expression of mUG/CCSP. In situ hybrid-ization analysis detected the mRNA for mUG/CCSP no earlier than on fetal day 17and, specifically, in the Clara cells. However, immunohistochemical analysis usingantibodies either to the rabbit UG/CCSP or to the mUG/CCSP protein could notdetect any expression until day 18.37

The mUG/CCSP cDNA was also used to clone the genomic sequences of themUG/CCSP gene. Three genomic clones ranging in size from 12 to 15 kb were iso-lated from a 129/SV mouse genomic library. The clone with the longest 5′-flankingsequences, that is, 5.1 kb, was characterized and 7.7 kb of this clone was se-quenced.38 The region sequenced included 3.3 kb of 5′-flanking DNA, the entirecoding region, and 0.2 kb of 3′-flanking DNA. The mUG/CCSP gene consists ofthree exons of 114, 187, and 125 bp separated by two introns of 2434 and 1373 bp.This gene structure is conserved between all species where the UG/CCSP gene hasbeen cloned, with differences in the sizes of the intervening sequences.39 The clon-ing of the gene for UG/CCSP has been important and has allowed for an extensiveanalysis of the molecular regulation of transcription of this gene.

REGULATION OF UG/CCSP GENE TRANSCRIPTION

The elements that regulate UG/CCSP gene expression in the lung have been ex-tensively studied in vitro by transient transfection analysis in a Clara cell–like adeno-carcinoma cell line, the H441 cells, and in vivo by transgenic mouse analysis.40–45

Transgenic mouse analysis demonstrated that 800 base pairs (bp) of the mUG/CCSPpromoter were sufficient for directing expression of a human growth hormone(hGH) reporter gene to the Clara cells of the lungs at a level comparable to that ofthe endogenous mUG/CCSP mRNA.43 Furthermore, a transgene containing a dele-tion of the mUG/CCSP promoter that contained only 166 bp of 5′-flanking DNA wasable to direct expression of the reporter gene to the lungs of transgenic mice. How-ever, the frequency and level of expression of the hGH reporter transgene were sig-nificantly reduced in this transgenic model. The level of expression of hGH underthe control of the proximal promoter region (166 bp) was, at maximum, 10% of thatof the endogenous mUG/CCSP gene. Therefore, it was demonstrated that there aretwo promoter regions in the mUG/CCSP 5′-flanking region: a proximal promoter re-gion located between −166 and the start of transcription and a distal promoter regionlocated between −800 and −166. The distal promoter region is responsible for 90%of mUG/CCSP gene expression. A diagram of the promoter elements for the mouseUG/CCSP gene is shown in FIGURE 1. Transient transfection, cotransfection, electro-phoretic mobility shift (EMS), and DNase 1 footprint analysis have identified thecis-acting elements that regulate transcription of the rat and mouse UG/CCSP gene.A summary of the elements postulated to regulate the UG/CCSP gene is illustratedin FIGURE 1. It has been demonstrated by mutation analysis and transfection analysisthat hepatocyte nuclear factors (HNF), HNF3α and HNF3β,40,44,45 AP-1,44 andNKx 2.142,46 are important for regulating basal transcription of UG/CCSP. Other


transcription factors indicating that there is indirect evidence for regulating UG/CCSP include SP1, SP3,47,48 and STAT1.29 However, no functional analyses of therole of these latter transcription factors have been demonstrated. Also, DNase 1 foot-print analysis has identified DNA-protein interactions at −236 to −21345 and at −314to −295.29 These elements in the distal promoter region of the UG/CCSP gene havenot been thoroughly investigated due to the limitation of available reagents to ana-lyze the UG/CCSP promoter.

MOUSE TRANSFORMED CLARA CELLS

The majority of the analysis of the UG/CCSP promoter has been conducted usingthe H441 cell line. The use of the H441 cell line may not be entirely appropriatebecause this cell line does not express the endogenous human UG/CCSP gene. Tran-sient transfection analysis has demonstrated that H441 cells do not recognize thecontribution of the distal promoter region of UG/CCSP toward UG/CCSP gene ex-pression.43 Identification of the elements in the distal promoter important for UG/CCSP expression has been accomplished by searches for sequence homology,cotransfection experiments in heterologous cell types, and transgenic analysis.42,45

Although this last approach has been used to identify NKx 2.1 as a potent distal pro-moter regulator of UG/CCSP, this approach is time-consuming, expensive, and notfeasible to investigate the interactions of elements in the distal promoter that are re-sponsible for the regulation of 90% of the expression of UG/CCSP. Thus, an attemptwas made to develop an alternative cell line that continued to express UG/CCSP andcould serve as a reagent for functional binding studies involving the UG/CCSP gene.This was accomplished by targeting the SV40 large T antigen (TAg) to the Claracells of mice and isolating transformed Clara cells that retained the expression of theendogenous mUG/CCSP gene.

Transgenic mouse lines were generated with the SV40 TAg under the control ofeither the rabbit or the mouse UG/CCSP promoter.47,48 Tumors were formed in miceexpressing each transgene and 10 clonal cell lines were derived from these tumors.The cell lines were named mTCC for mouse transformed Clara cells.29 The mTCClines exhibited an epithelial phenotype in culture. Immunohistochemical, Northernblot, and Western blot analyses were used to characterize UG/CCSP expression inthese cells. All clones isolated expressed the UG/CCSP gene. However, the level ofexpression of UG/CCSP in these cell lines was reduced to 0.7% of that found in totallung mRNA. The reduction in UG/CCSP expression was limited not only in the

FIGURE 1. A schematic representation of the proximal and distal promoter regions ofUG/CCSP. Hatched bars indicate a region where DNA/protein interaction has been ob-served, but no transcription factor has been identified.


mTCC cell lines, but also in the tumors in situ. The cell lines were analyzed byNorthern blot analysis for their ability to express other lung-specific differentiationmarkers. These cells expressed SP-B, NKx 2.1, HNF3α, and HNF3β, but did not ex-press SP-A, SP-C, or SP-D. Interestingly, unlike H441 cells, these cells expressedUG/CCSP and the transcription factor HNF3. The expression of the HNF3β proteinis important if these cells are to be used in defining the regulation of NKx 2.1 geneexpression.

UTILITY OF MTCC LINES

The usefulness of the mouse transformed Clara cells to investigate the molecularregulation of UG/CCSP was made apparent when an investigation was initiated intothe potential regulation of UG/CCSP by cytokines. UG/CCSP has been recentlyshown to inhibit both the action and expression of IFN-γ in the lungs.15 If the phys-iological role of UG/CCSP is to regulate IFN-γ, then a negative feedback regulatoryloop may exist between UG/CCSP and IFN-γ. In this loop, IFN-γ would stimulateUG/CCSP expression and, in turn, UG/CCSP would inhibit IFN-γ. Using the mTCClines, it was shown that IFN-γ stimulated the expression of UG/CCSP mRNA inthese cells. This regulation of UG/CCSP by IFN-γ was verified in vivo when a sim-ilar induction of CC10 mRNA was observed after intratracheal administration ofIFN-γ to mouse lungs.29

POTENTIAL MECHANISMS OF CYTOKINE REGULATIONOF UG/CCSP

Examination of the promoter elements identified to date shows several pointswhere cytokines can regulate the expression of UG/CCSP. IFN-γ has been shown toregulate UG/CCSP expression in both the proximal and distal promoter regions.29

IFN-γ activates gene expression by binding to its receptor located in the cell plasmamembrane. Ligand binding of IFN-γ to its receptor triggers the activation of theJanus kinase (JAK)–signal transducer and activator of transcription (STAT) pathway.Activation of the JAK-STAT pathway can activate gene expression by two routes.First, IFN-γ activation of the STAT protein will cause STAT1 to bind to a gammaactivation sequence (GAS) in the promoter region of the gene and will activate tran-scription.49–53 Second, activation of this JAK-STAT pathway can induce the synthe-sis of another transcription factor, the interferon response factor, IRF-1, which canactivate gene transcription by binding its own response element in the promoter re-gion of a gene.54 Both pathways are possible for the regulation of mUG/CCSP geneexpression. A GAS site has been located by footprint analysis in the distal promoterregion of the UG/CCSP promoter.29 Also, it has been demonstrated that HNF3β isregulated by IFN-γ through activation of the IRF pathway.55 Therefore, cytokineshave the potential to activate UG/CCSP gene expression by signaling through theGAS site in the distal promoter of UG/CCSP and/or by stimulating through HNF3βactivity on the proximal promoter. A summary of the potential regulatory pathwaysfor UG/CCSP by cytokines is shown in FIGURE 2.


POTENTIAL MECHANISMS OF O2 REPRESSION OF UG/CCSP

Examination of the promoter elements of mUG/CCSP also shows two potentialmechanisms for O2 repression of the mUG/CCSP promoter based on how hyperoxiaregulates the activity of known transcription factors. Oxygen has been shown to reg-ulate gene expression of antioxidant response enzymes by activating a signaling cas-cade that results in the activation of transcription factors, such as nuclear factorkappa B (NFκB) and AP-1. These factors bind their response element and activategene transcription.56–59 It has been shown that AP-1 binds the UG/CCSP promoter,making it a prime candidate for mediating O2 regulation of UG/CCSP.44 AP-1 is acomplex of two families of transcription factors: the cJun and cFos families. ThecJun family consists of vJun, cJun, JunB, and JunD. The cFos family consists ofvFos, cFos, FosB, Fra-1, and Fra-2.60 Examination of the composition of AP-1 inH441 cells has demonstrated that the AP-1 complex that binds to the UG/CCSP pro-moter consists of a heterodimer of the transcription factors JunB and Fra-2.44 WhileJun-Fos heterodimers activate gene transcription, Fra-JunB heterodimers do not.60

Also, the AP-1 site in the UG/CCSP promoter overlaps with one of the HNF3β bind-ing sites. It has been shown that AP-1 and HNF3β cannot simultaneously occupy thesame site in the UG/CCSP promoter.44 Therefore, one possible mechanism for O2repression of UG/CCSP gene expression is that O2 regulation of UG/CCSP gene ex-pression induces AP-1 to the site and subsequent competition of AP-1 with HNF3βon the UG/CCSP promoter.

The second possible mechanism of O2 repression of UG/CCSP may involve O2impairing the activity of NKx 2.1 to activate UG/CCSP gene transcription. It hasbeen shown that oxidation of NKx 2.1 causes the formation of concatamers of this

FIGURE 2. The potential mechanisms of IFN-γ stimulation of UC/CCSP expression.(A) One potential mechanism is IFN-γ activation of STAT1, which binds directly to the GASsite in the distal promoter of the UG/CCSP gene. (B) A second possible mechanism for IFN-γ stimulation is the indirect increase in HNF3β levels after STAT activation throughincreased transcription of HNF3β by IRF-1.


transcription factor.61 Multimers of NKx 2.1 do not bind DNA as well as monomersand therefore are not as transcriptionally active. If O2 causes oxidation of NKx 2.1,then this may be a possible mechanism of O2 repression of UG/CCSP. Although O2inactivation of NKx 2.1 is a possible mechanism, it is not likely. While hyperoxiarepresses UG/CCSP gene expression, transcription of surfactant proteins A, B, andC is elevated.31,35 Since the transcription of the surfactant protein genes is also de-pendent on NKx 2.1,62 O2 repression of NKx 2.1 activity would result in the repres-sion of these genes and not activation. A summary of these potential mechanisms isshown in FIGURE 3.

FIGURE 3. The potential mechanisms of hyperoxic repression of UG/CCSP gene ex-pression. (A) Oxygen stimulates AP-1 binding to the UG/CCSP proximal promoter region,which displaces the HNF3β binding. (B) Oxygen causes NKx 2.1 to no longer bind the UG/CCSP promoter.


DISCUSSION

Although the precise physiological significance of UG/CCSP expression andsecretion has not been totally defined, this molecule has served as a marker for theinvestigation of the cell-specific and physiological regulation of pulmonary geneexpression. Identification of the UG/CCSP promoter has identified two promoterregions regulating gene expression. Current analysis of these promoter regions hasdemonstrated that the distal promoter region is responsible for regulating 90% of thisgene activity in vivo. However, the proximal promoter regions are important forgoverning the cell-specific regulation of this gene. These promoter elements areanalogous to the circuitry of a radio, with the proximal promoter being the tuner andthe distal promoter region being the amplifier (FIG. 4). Current investigations haveidentified the transcription factors found to be important in regulating UG/CCSPgene expression. These investigations have demonstrated that lung-specific expres-sion of UG/CCSP is not due to the presence of lung-specific transcription factor(s),but due to the appropriate combination of non-tissue-specific transcription factors.However, these studies have not identified elements responsible for the preferenceof the UG/CCSP gene to be expressed in the Clara cells. Many, if not all, of the ele-ments identified to be important for UG/CCSP gene expression have been demon-strated to be expressed in the alveolar type II cell. Future studies must be conductedto increase our understanding of how these elements coordinate the cell-specificpreference of the UG/CCSP promoter.

ACKNOWLEDGMENTS

This manuscript was produced with the technical help of John Ellsworth and theeditorial assistance of Janet DeMayo. This work was supported by NIH Grant No.HL61406.

FIGURE 4. A schematic representation of the functional regions of the UG/CCSP pro-moter. The distal promoter regulates 90% of the transcriptional activity and is portrayed asan amplifier (triangle). The proximal promoter is important for cell-specific expression ofUG/CCSP and, possibly, a key regulatory point is represented as a tuner.


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Physiological Regulation of Uteroglobin/CCSP Expression

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