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Molecular Aspects of Medicine 28 (2007) 453–480

Review

Tocopherol transfer protein deficiency modifiesnuclear receptor transcriptional networks

in lungs: Modulation by cigarette smoke in vivo

K. Gohil *, S. Oommen, V.T. Vasu, H.H. Aung, C.E. Cross

Pulmonary and Critical Care Medicine, Genome and Biomedical Sciences Facility,

451 East Health Sciences Drive, University of California, Davis, CA 95616, USA

Received 2 February 2007; revised 12 February 2007; accepted 13 February 2007

Abstract

Dietary factors and environmental pollutants initiate signaling cascades that converge onAhR:Nrf2:NF-jB transcription factor (TF) networks and, in turn, affect the health of theorganism through its effects on the expression of numerous genes. Reactive oxygen metabolites(ROMs) have been hypothesized to be common mediators in these pathways. a-Tocopherol(AT) is a potent, lipophilic, scavenger of ROMs in vitro and has been hypothesized to be a majorchain-breaking anti-oxidant in lipoproteins and biological membranes in vivo. The lung offers avital organ to test the various postulated actions of AT in vivo. Lung AT concentrations can bemanipulated by several methods that include dietary and genetic techniques. In this study wehave used mice with severe AT deficiency inflicted at birth by the deletion of AT transfer protein(ATTP) which is abundantly expressed in the liver and regulates systemic concentrations of AT.Mice and humans deficient in ATTP are AT deficient. Female ATTP-deficient (ATTP-KO)mice and their congenic ATTP normal (WT) mice fed a diet containing 35 IU AT/kg diet wereused to test our hypothesis. The mice (n = 5/group) were exposed to either air or cigarettesmoke (CS, total suspended particles 60 mg/m3, 6 h/day), a source of ROM, for 3 or 10 days.Post-exposure lung tissue was dissected, RNA extracted from each lung and it was pooledgroup-wise and processed for GeneChip analysis (Affymetrix 430A 2.0). Differential analysisof the transcriptomes (�16,000 mRNAs) identified CS sensitive genes that were modulatedby lung AT-concentration. CS activated AhR driven genes such as cyp1b1 whose induction

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* Corresponding author. Tel.: +1 530 754 6932; fax: +1 530 752 8632.E-mail address: kgohil@ucdavis.edu (K. Gohil).

454 K. Gohil et al. / Molecular Aspects of Medicine 28 (2007) 453–480

was augmented in CS-exposed, AT-deficient lungs. However, CS-induced expression of some ofthe Nrf2 driven genes was not potentiated in the AT-deficient lungs. Largest clusters of CS-ATsensitive genes were lymphocyte and leukocyte specific genes. These gene-clusters includedthose encoding cytokines and immunoglobulins, which were repressed by CS and were modu-lated by lung AT concentrations. Our genome-wide analysis suggests reciprocal regulation ofxenobiotic and immune response genes by CS and a modulatory role of lung AT concentrationon the expression of these clusters of genes. These data suggest that in vivo network of AT, AT-metabolites and ATTP affects the transcription of genes driven by AhR, Nrf2 and NF-jB, tran-scription factor networks that transduce cellular metabolic signals and orchestrate adaptiveresponses of lungs to inhaled environmental pollutants.� 2007 Elsevier Ltd. All rights reserved.

Keywords: Cigarette smoke; Tobacco; d-Chip; Gene-networks; Immune response; Inflammation;Microarrays; a-Tocopherol transfer protein; Arntl; Nrf2; NF-jB; Vitamin E

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 455

1.1. Lessons from yeast: transcription of multiple genes is affected by nutrients

and oxidant products of metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . 4551.2. Genome-wide responses to AT in vivo . . . . . . . . . . . . . . . . . . . . . . . . . 456

1.2.1. Transcriptional responses to dietary AT . . . . . . . . . . . . . . . . . . . . 4561.2.2. Transcriptional responses to AT due to deletion of ATTP-gene . . . 4571.2.3. Transcriptional responses of lungs to inhaled oxidants and

xenobiotics of cigarette smoke (CS) . . . . . . . . . . . . . . . . . . . . . . . 4572. Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 458

2.1. ATTP-KO and WT mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4582.2. CS exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4592.3. RNA extraction and, GeneChip, quantitative real-time PCR (qRTPCR)

and immunoblot analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4592.4. Statistical data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 459

3. Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 459

3.1. ATTP-KO mice do not express ATTP in liver or lungs . . . . . . . . . . . . . 4593.2. Genome-wide responses of lungs to AT-deficiency and to CS . . . . . . . . . 460

3.2.1. Genome-wide responses of lungs to AT deficiency in the absenceof ‘‘oxidative–xenobiotic’’ stress . . . . . . . . . . . . . . . . . . . . . . . . . . 460

3.2.2. Genome-wide responses of lungs to oxidative and xenobiotic stressof CS in AT deficient lungs of ATTP-KO mice . . . . . . . . . . . . . . . 463

3.3. Induction of AhR driven genes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4663.4. Modulation of Nrf2 driven genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4693.5. Modulation of immune-inflammatory genes. . . . . . . . . . . . . . . . . . . . . . 470

4. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 473Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 474References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 474

K. Gohil et al. / Molecular Aspects of Medicine 28 (2007) 453–480 455

1. Introduction

1.1. Lessons from yeast: transcription of multiple genes is affected by nutrients and

oxidant products of metabolism

The invention and application of DNA and oligonucleotide microarray tools toquantify genome-wide expression of mRNAs have demonstrated that a single nutri-ent can generate complex transcriptional responses. The utility and power of thesetools to define transcriptional networks operating in vivo is exemplified by studiesin yeast (DeRisi et al., 1997; Lipshutz et al., 1995; Stengele et al., 2005). These studiesoffer a paradigm for investigations into defining the diversity of the actions of vita-min E in vivo. The yeast (Saccharomyces cerevisiae) genome is distributed over 16chromosomes containing 5654 predicted protein coding genes (David et al., 2006).In yeast, repletion of glucose initiates a major transcriptional reprogramming inresponse to extracellular glucose (DeRisi et al., 1997; Kresnowati et al., 2006; West-ergaard et al., 2006). The signaling cascade initiated by a change in extracellular glu-cose concentration converges on distinct domains of the genome and RNApolymerase complex containing �150 proteins to initiate the glucose sensitive tran-scriptional program. The changes in the transcription of most of the glucose sensitivegenes can be accounted for by coordinated interactions of a few transcription factors(TFs), TF-network, on their specific targets on the genome. For example, glucosesensitive activation of 589 genes of the yeast genome could be accounted for bythe activities of 12 TFs (Kresnowati et al., 2006). Although such comprehensivestudies on the action of glucose in mammalian cells are lacking, genome-wideresponses of b-pancreatic cells to glucose suggest that a large number of genes aremodulated in response to glucose (Schuit et al., 2002); functional classification ofthe affected genes led the authors to suggest that glucose stimulated the conversionof mitochondrial metabolites into lipid intermediates. Transcriptional mechanismsthat may co-ordinate glucose and lipid metabolism remain to be characterized andinclude recently discovered TFs such as carbohydrate responsive element bindingprotein (Dentin et al., 2006). It is likely that mammalian cells will have distinct cellspecific, and yet to be defined, transcriptional responses to vital nutrients such as glu-cose and vitamins such as AT.

Aerobic metabolism of glucose and other nutrients generates reactive oxygenmetabolites (ROMs) such as hydrogen peroxide (H2O2) which is used as a molecularweapon to kill invading microbial pathogens (El-Benna et al., 2005), as a molecularsignal that affects cell cycle and survival (Stone and Yang, 2006), and as a mediatorof tissue damage (Bergamini et al., 2004; Frisard and Ravussin, 2006; Jaeschke,2006; Xu and Touyz, 2006). Search for genome-wide transcriptional signatures ofchanges in H2O2 concentrations have identified a complex response of a genometo H2O2. The expression of �900 yeast genes are simultaneously affected by H2O2

and other oxidants (Gasch et al., 2000). Similarly, exposure of murine macrophagesto H2O2 (Zhang et al., 2005) affected the expression of 113 genes that regulate cellsurvival, stress, and metabolism of glucose and lipids. The H2O2 stress also affectedthe activities of several TFs such as NF-jB, p53 and Akt. These observations and

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those from other studies show that a single oxidant species can affect the expressionof a genome by altering the expression numerous genes that are compartmentalizedin distinct regions of the genome.

1.2. Genome-wide responses to AT in vivo

Descriptions of vitamin E deficiency symptoms suggest that this micronutrientmust have complex effects on the expression of the genome during the mammal’s life-span, particularly during conception and early development. The two well identifiedeffects of vitamin E deficiency are infertility and neuromuscular disease. More than80 years after the discovery of the deficiency symptoms, transcriptional signatures ofvitamin E for the prevention of infertility are lacking and remain a major deficiencyin our knowledge of the molecular actions of AT, the most abundant member ofvitamin E family in mammalian tissues. The need for dietary AT for fetal growthmay not be unique to rodents as has been suggested by a recent study in humans(Scholl et al., 2006).

1.2.1. Transcriptional responses to dietary AT

We are beginning to witness the complexity of the in vivo effects of AT on the neu-romuscular and the central nervous systems. Recent genome-wide analyses ofmRNAs extracted from muscle and from brain suggest multiple molecular targetsthat may participate in neuromuscular disease caused by chronic AT deficiencyin vivo inflicted by dietary AT-deficiency. In male rats dietary AT-deficiency for�3 months activated 56 muscle genes (Nier et al., 2006); functional classificationof the affected genes identified those encoding anti-oxidative, anti-inflammatory,anti-fibrotic and, muscle and extracellular matrix proteins.

The transcriptional response to chronic dietary AT deficiency also affects the tran-scriptomes of the cerebral cortex (Hyland et al., 2006) and the hippocampus (Rotaet al., 2005). Dietary AT-deficiency for 14 months in male rats resulted in the repres-sion of 34 genes that included genes encoding myelin proteins and those for neuronalsignal propagation, suggesting molecular targets that may account for neurologicaland electrophysiological alterations (Hyland et al., 2006). AT-sensitive cortical genesalso included 11 genes that were induced by AT deficiency and included those encod-ing catalase and tenascin-R. Nine months of dietary AT-deficiency in male ratsaffected the transcription of diverse functional clusters of genes that included hor-mone and hormone metabolism, neuronal survival, dopaminergic system and amy-loid proteins in the rat hippocampus (Rota et al., 2005).

Dietary AT deficiency also affected the expression of testes genome (Barellaet al., 2004b; Rota et al., 2004) and that of the liver (Barella et al., 2004a) andlungs (Oommen et al., 2007). The latter study identified a coordinated regulationof a cluster of �13 cytoskeleton genes that appear to be regulated by serumresponse factor, a transcription factor important in the development of myocar-dium and skeletal muscle (Miano, 2003). Genome-wide screens of mRNA expres-sion of T-cells isolated from mice fed different AT-containing diets suggest thatAT affects cell cycle and Th1/Th2 balance (Han et al., 2006). These genome-wide

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screens of mRNA expression in vivo suggest that AT has system-wide effects ontissue specific transcriptomes.

1.2.2. Transcriptional responses to AT due to deletion of ATTP-gene

The discovery of patients with systemic AT-deficiency in spite of ingesting a dietcontaining normal amounts of vitamin E has highlighted the physiological require-ment of AT for the health of the neurological system (Mariotti et al., 2004; Ouahchiet al., 1995) in humans. The identification of mutations in the ATTP gene which isprimarily expressed in the liver has emphasized the importance of a hepatic proteinin determining neuromuscular health. Two research groups independently developedtransgenic mice lacking ATTP (Jishage et al., 2001; Terasawa et al., 2000). In ATTP-KO mice the extrahepatic AT concentrations are <10% of the ATTP-normal (WT)mice and they develop ataxia with aging in spite of ingesting a standard rodent dietcontaining abundant AT (Yokota et al., 2001). Therefore, the ATTP-KO miceappear to recapitulate the biochemical and behavioral phenotype of patients withATTP mutations and offer an in vivo model to investigate AT sensitive transcripto-mes that may be relevant to the human disease.

System-wide effects of AT on tissue specific transcriptomes have been reported inATTP-KO mice that were fed a standard AT diet (Gohil et al., 2004); the study sug-gested that hearts were least sensitive to AT-ATTP deficiency (�50 genes affected)and adrenal glands were most sensitive to AT-ATTP deficiency (�2000 genesaffected). Global mRNA expression analysis of cerebral cortex from young ATTP-KO mice suggests that AT is required for myelination, synaptogenesis and formationof functional axons (Gohil et al., 2003b). The possible role of ROM in the regulationof these cortical genes in not known and at present the actions of AT appear to sug-gest a non-antioxidant action of AT. The genome-wide mRNA screen also identifiedthe induction of a number of genes that had been purported to be induced by ROM.However, the results were also remarkable for the lack of a significant change in theexpression of genes such as heme-oxygenase-1 and glutamate-cysteine ligase that areclassically associated with increased ROM production.

Some of the apparently non-antioxidant actions of AT were hypothesized to bemediated by a retinoic acid related orphan receptor-a (RORa) (Gohil et al., 2004,2003b) which is a member of nuclear receptor superfamily and is a transcription fac-tor that affects the expression of a large number of genes that include genes for lipidmetabolism (Lau et al., 2004). Intriguingly, mice with mutations in or in the deletionof RORa gene develop ataxia (Jetten et al., 2001) suggesting that the neurologicalphenotype of ATTP-KO mice and those of patients with ATTP mutations may bemediated through RORa.

1.2.3. Transcriptional responses of lungs to inhaled oxidants and xenobiotics of

cigarette smoke (CS)

The lung offers a vital organ to test, in vivo, the relative contribution of non-anti-oxidant and antioxidant actions of AT. Oxidative and nitrosative stresses are fre-quently invoked as causative or propagative mechanisms of lung injury(Andreadis et al., 2003; Haddad, 2002; Mossman et al., 2006; Rahman and Kilty,

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2006; van der Vliet et al., 2000). It is thus reasonable to hypothesize that modulationof the antioxidant capacity of the lung should modulate oxidant-induced lung injury.We and others (Engelhardt, 1999) are engaged in testing this hypothesis in mice. Weare specifically interested in defining transcriptional targets of oxidant-antioxidantactions in mice because the putative targets from the mouse genome are amenablefor testing cause-effect relationships in vivo. In addition, mice can be exposed to oxi-dant stress of ozone (Gohil et al., 2003a) or cigarette smoke (Meng et al., 2006; Wit-schi et al., 2004) under conditions that can be manipulated to test specific hypothesisin the laboratory. We recognize that intervention trials with antioxidants in humanshave not shown beneficial effects (Aitio, 2006; Czernichow and Hercberg, 2001; Dan-gour et al., 2004; Welty, 2001). We speculate that further studies in mice may providecellular and molecular basis for these failures and assist in designing intervention tri-als that are based on in vivo actions of lipid soluble micronutrients that have antiox-idant properties in vitro.

Genome wide screens of mRNA expression can prove useful in screening theeffects of environmental pollutants (Leikauf et al., 2001). Our analysis of lung tran-scriptome of mice exposed to ozone identified a cachexia-like transcriptional signa-ture in lungs that appeared to be driven by NF-jB pathway (Gohil et al., 2003a).Our more recent studies in ATTP-KO mice suggest a similar recruitment ofNF-jB pathway in the lungs of ozone breathing mice (Vasu et al. manuscript in prep-aration). Responses of mouse lung transcriptomes to CS show changes in the expres-sion of hundreds of genes (Gebel et al., 2006; Meng et al., 2006; Rangasamy et al.,2004). Functional classification of the affected genes shows the recruitment of diversebiological processes such as phases I and II responses, inflammation and cell sur-vival. Bioinformatic analysis of the CS sensitive lung transcriptome suggests thatthe molecular signaling pathways triggered by CS converge on at least three ubiqui-tous transcription factors; aromatic hydrocarbon receptor (AhR) (Gebremichaelet al., 1996; Villard et al., 1998), nuclear factor erythroid 2-related factor 2 (Nrf2)(Rangasamy et al., 2004), and nuclear factor j of B cells (NF-jB). A number ofreports suggest that ROMs play an important role in the activation of Nrf2 (Choet al., 2006; Kensler et al., 2006) and NF-jB (Rahman et al., 2004; Wang et al.,2002). Based on these observations we hypothesized that acute CS exposure ofATTP-KO mice which have severe AT deficiency in lungs (AT < 10% of WT mice)will show augmented responses of genes regulated by Nrf2:NF-jB transcription fac-tor network. This hypothesis was tested in mice exposed to CS for up to 10 days fol-lowed by transcriptomic analysis of lung tissue.

2. Methods

2.1. ATTP-KO and WT mice

The ATTP-KO mice and their congenic WT ‘‘siblings’’ were obtained from breed-ing pairs of heterozygous mice. A major advantage of this breeding strategy is thatthe ATTP-KO and the WT controls have identical genetic background, and housing

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and dietary conditions, factors that eliminate the contributions of these variables inthe interpretations of gene expression data. Female mice were used in this study andwere allowed to feed on a semi-synthetic diet that contained 35 IU of tocophorol ace-tate/kg diet (Oommen et al., 2007).

2.2. CS exposure

Five months old female mice of the two genotypes were exposed to either air orCS for either 3 or 10 days as previously described (Witschi et al., 2004) except thatCS contained 60 mg/m3 of total suspended particles. Each treatment group con-tained 5 mice. The mice were euthanized within 2 h after the last CS exposure. Airbreathing mice were also euthanized on the same day. Whole blood was collectedby cardiac puncture and lungs were rapidly dissected free of large blood vesselsand airways and frozen on dry-ice and then stored at minus 70 �C until RNA extrac-tion (within 4 weeks).

2.3. RNA extraction and, GeneChip, quantitative real-time PCR (qRTPCR) and

immunoblot analyses

These procedures were performed without any modification as previouslydescribed (Oommen et al., 2007).

2.4. Statistical analysis of data

Fold-changes in gene expression obtained by qRTPCR were analyzed by Graph-Pad PRISM (version 4.0; GraphPad Software, San Diego, CA). An unpaired Stu-dent’s t test was used for comparisons between the treatments. All data wereconsidered statistically significant when P values were 60.05. The data are reportedas means ± SEMs.

3. Results and discussion

3.1. ATTP-KO mice do not express ATTP in liver or lungs

Immunoblot analysis of lung and liver homogenates with anti-ATTP antiserumshowed a �32 kDa protein, abundantly expressed in WT liver but undetectable inATTP-KO liver, or in lungs from WT or ATTP-KO mice (Fig. 1). Previous stud-ies have shown that the lungs and plasma of ATTP-KO mice have <10% AT con-centrations of WT plasma and lungs (Leonard et al., 2002; Schock et al., 2004).Collectively, these data show that the deletion of ATTP-gene results in very lowAT (<10% of WT) in the plasma and lungs of mice fed a standard rodent dietcontaining 35 IU AT/kg diet. AT-deficiency achieved by gene deletion is similarto that achieved by feeding an AT-depleted diet for 5 months (Oommen et al.,2007).

Fig. 1. ATTP is expressed in WT-livers but it is not detectable in ATTP-KO livers or in lungs from eitherWT or ATTP-KO mice. Immunoblot analyses of liver and lung homogenates. Rabbit anti-ATTPantiserum (a generous gift from Professor Maret G.Traber) detected �32 kDa ATTP in liver homogenatesfrom the two WT mice (lanes 1 and 2) but not in the liver homogenates from the two ATTP-KO mice(lanes 3 and 4) or from lung homogenates of the two WT mice (lanes 1 and 2) or the two ATTP-KO mice(lanes 3 and 4). Decreased expression of the higher molecular weight protein in liver homogenates fromone of the two ATTP-KO mice (lane 4 for liver) compared to that in the liver homogenates from the twoWT mice or the ATTP-KO mouse may be attributed to biological variation or decreased expression of theprotein which appears to cross-react with the anti-TTP antiserum or due to analytical artifacts.

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3.2. Genome-wide responses of lungs to AT-deficiency and to CS

The most recent estimate of protein coding genes in the mouse genome is 56,722(Maeda et al., 2006). This estimate offers the maximum number of genes that couldbe modulated by AT. Utilization of mouse genome arrays containing oligonucleotideprobes for �22,000 genes for screening the expression of mRNAs prepared fromlung homogenate identified the expression of �16,000 genes (see below Table 4).The total number of expressed genes appears to be very similar in the lungs fromWT and from ATTP-KO mice. The deletion of the ATTP gene and the resultingdepletion of plasma and lung AT affected the expression of �2125 genes that weredistributed over all of the 21 chromosomes (Fig. 2). The data indicate that AT sen-sitive genes are present on all the chromosomes whose genes are represented onGeneChips. The mechanisms by which AT may simultaneously regulate the expres-sion of genes that are physically separated over long stretches of DNA–protein com-plexes across the various chromosomes remain unresolved.

3.2.1. Genome-wide responses of lungs to AT deficiency in the absence of ‘‘oxidative–

xenobiotic’’ stress

ATTP deficiency resulted in a large induction of the lung transcriptome (Fig. 2,inset). In addition to the induction of genes that respond to cellular stress such as‘‘redox-stress’’ predicted from the postulated antioxidant actions of AT, severalgenes that play an essential role in responding to xenobiotic challenge were alsoinduced (Table 1). These included genes that encode proteins in the AhR pathwayand the redox sensitive Nrf2-driven pathway which has been proposed to ‘‘defendlung from oxidative stress’’ (Cho et al., 2006). A sixfold induction of mRNA encod-ing the kelch-like ECH-associated protein 1 (Keap1) in AT deficient lung is partic-

Fig. 2. Chromosomal localization of AT sensitive genes in lung tissue. All the AT-sensitive genes whosechromosomal location was known were expressed as % of total number of genes on the GeneChip fromthe respective chromosome. The data suggest that AT-deficiency had no selective effect on the expressionof genes from any specific chromosome. The inset shows that more genes were activated then repressed.

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ularly noteworthy (Table 1). Keap1 is a zinc-metalloprotein that has two cysteineresidues that have been proposed to respond to changes in intracellular redox state(Dinkova-Kostova et al., 2002; Dinkova-Kostova et al., 2005; Levonen et al., 2004).The precise mechanism(s) by which the Keap1 gene is transcribed is not fully char-acterized. Our data suggest that AT-deficiency increases mRNA encoding Keap1, aprotein that is suggested to be sensitive to redox-state of a cell. Since Keap1 regulatestranscriptional activity of Nrf2 (Kobayashi et al., 2006), our data also suggest that anumber of genes modulated by lung AT-deficiency are candidate Nrf2-driven genesin vivo in lungs.

AT-deficiency also resulted in the induction of a large cluster of genes that encodemembers of the nuclear receptor superfamily (Table 2). Nuclear receptors (NRs) areDNA-binding transcription factors that regulate the expression of a large number ofgenes that affect a broad spectra of physiological functions (Germain et al., 2006).Co-ordinated induction of this cluster of genes identifies novel transcriptional targetsof AT or AT induced changes in metabolites in lung tissues. The co-ordinated induc-tion of peroxisome proliferator activator receptor-a (Ppara) and nuclear receptorco-repressor and co-activator suggests a concerted effect of AT-deficiency on a tran-scriptional pathway that is implicated in the regulation of important cellular func-tions (Feige et al., 2006). The genes encoding Ppara and Pparbp, and othermembers of this family of genes, are important in cholesterol and fatty acid metab-olism (Kim et al., 2003; Li and Glass, 2004). A recent study has implicated anothermember of NR family, Pparc, to be a mediator of AT action on lipoprotein inducedexpression of monocyte CD36 (Munteanu et al., 2006). Table 3 lists some of thegenes important in lipid metabolism that were induced in the lungs of ATTP-KO

Table 1ATTP-deficiency activates transcription of phase I, phase II and stress response genes

Gene title Gene symbol Signal intensity Fold change Probe set ID

WT KO

Kelch-like ECH-associatedprotein 1

Keap1 60.1 397.3 6 1421721_a_at

Aryl-hydrocarbon receptor Ahr 17.6 209.4 9 1450695_atAryl hydrocarbon receptor

nuclear translocatorArnt 98.4 182.5 3 1450747_at

Heme oxygenase (decycling) 1 Hmox1 275.8 1459.9 5 1448239_atGlutamate-cysteine ligase,

catalytic subunitGclc 1158.4 4335.3 4 1424296_at

Cytochrome P450, family 1a1 Cyp1a1 124.7 300 2 1422217_a_atCytochrome P450, family 2,

subfamily a4Cyp2a4 1119.8 2631.9 2 1422230_s_at

Glutamate-cysteine ligase,modifier subunit

Gclm 479.4 1069.6 2 1418627_at

Alcohol dehydrogenase7 (class IV)

Adh7 239.8 449.5 2 1450110_at

Superoxide dismutase 2,mitochondrial

Sod2 22.2 67 3 1417194_at

Superoxide dismutase 3,extracellular

Sod3 621.1 1417.1 3 1417634_at

Heat shock protein 1A Hspa1a 592.9 4761 7 1452388_atHeat shock protein 8 Hspb8 172 818 5 1417014_atHeat shock protein

90kDa alphaHsp90aa1 1053.8 5215.1 5 1437497_a_at

Heat shock protein 1B Hspa1b 1726.8 6358.2 4 1427127_x_atThioredoxin reductase 1 Txnrd1 44.5 242.6 4 1424486_a_atThioredoxin interacting protein Txnip 1118.2 3643.3 2 1415997_atHeat shock protein family,

member 7Hspb7 111.7 262.4 2 1434927_at

Ribosomal protein L18 Rpl18 5114 5939 1 1450372_a_atRibosomal protein L26 Rpl26 10,826 12,192 1 1436995_a_at

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mice. Collectively, these data from air breathing mice suggest that the lungs ofATTP-KO mice are under cellular stress, and may have adaptively modified lipidtransport and metabolism through AT’s actions on the expression and activities ofselect members of the nuclear receptor superfamily. A recent gene-profiling studyin livers of mice with a blunted expression of Ppara identified a cluster of 130 co-reg-ulated genes that were members of ubiquitin dependent protein catabolism pathways(De Souza et al., 2006). The data in Table 2, combined with the observations ofPpara deficient mice, prompted the search for a similar cluster in the AT sensitivegene profile. Data in Table 4 show 20 genes of the ubiquitin pathway that wereco-ordinately activated in the lungs of five months old female ATTP-KO mice; thesegenes included major regulators of the protein degradation pathway by the ubiquitinsystem (Hershko and Ciechanover, 1998) suggesting a role for AT in intracellularprotein catabolism. Collectively, these observations, and the putative mechanismsthat may modulate these pathways are illustrated in Fig. 3.

Table 2ATTP-deficieny activates members of nuclear receptor superfamily

Gene title Genesymbol

Signal intensity Foldinduction

Probe set ID

WT ATTP-KO

Nuclear receptor subfamily 1,group D, member 2

Nr1d2 54 625 12 1416959_at

Peroxisome proliferator activatedreceptor binding protein

Pparbp 149 1094 10 1421907_at

Aryl-hydrocarbon receptor Ahr 18 209 10 1450695_atNuclear receptor co-repressor 1 Ncor1 143 1551 10 1423201_atNuclear factor I/C Nfic 39 257 7 1422565_s_atNuclear factor of activated T-cells,

cytoplasmic, calcineurin-dependent 3Nfatc3 49 309 6 1452497_a_at

Nuclear receptor subfamily 2,group C, member 2

Nr2c2 24 160 6 1425014_at

Nuclear factor I/A Nfia 6 39 5 1427733_a_atNuclear receptor interacting

protein 1Nrip1 156 863 5 1449089_at

Nuclear receptor coactivator6 interacting protein

Ncoa6ip 100 309 4 1450400_at

Nuclear receptor subfamily 2,group F, member 2

Nr2f2 359 1339 4 1416159_at

Peroxisome proliferator activatedreceptor alpha

Ppara 21 92 3 1449051_at

Retinoic acid receptor, alpha Rara 36 166 4 1450180_a_atRetinoic acid receptor, gamma Rarg 89.9 368.1 3 1419416_a_atRetinoid X receptor alpha Rxra 56.2 203.5 2 1425762_a_atRAR-related orphan receptor alpha Rora 22.1 83.7 2 1424035_at

Ribosomal protein L18 Rpl18 5114 5939 1 1450372_a_atRibosomal protein L26 Rpl26 10,826 12,192 1 1436995_a_at

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3.2.2. Genome-wide responses of lungs to oxidative and xenobiotic stress of CS in AT

deficient lungs of ATTP-KO mice

Cigarettes contain a complex mixture of phytochemicals, synthetic molecules andminerals, and when ignited generate at least 4000 chemicals of diverse chemical reac-tivities and bioactivities (Borgerding and Klus, 2005; Counts et al., 2006). CS is esti-mated to contain 5 · 1014 free radicals per puff not including NO (Pryor et al., 1983).Inhaled CS-free radicals such as ROM/reactive nitrogen metabolites (RNM) areexpected to perturb the cellular redox systems such as the GSH-GSSG couple (Rah-man and MacNee, 1999) and possibly ascorbate-AT-b-carotene (BC) systems(Alberg, 2002) in various lung compartments and cause lung pathologies. Interven-tion strategies designed to bolster such redox systems by increasing dietary concen-trations of N-acetyl-cysteine (De Flora et al., 2003; Gillissen and Nowak, 1998) ormicronutrients such as AT and BC that extend over more than half a century ofresearch in animal models and in humans suggest complex interactions betweenCS, diet and genetic make-up of the organism and, a need for better understandingof the molecular and cellular responses of the lung in vivo.

Table 3ATTP-deficiency activates genes that regulate lipid metabolism

Gene title Genesymbol

Signal intensity Foldinduction

Probe set ID

WT ATTP-KO

Phosphodiesterase 4D interactingprotein (myomegalin)

Pde4dip 3 63 18 1460426_at

Lipase, endothelial Lipg 4 66 17 1421261_atProstaglandin–endoperoxide synthase 2 Ptgs2 52 575 13 1417263_at24-Dehydrocholesterol reductase Dhcr24 9 114 12 1418129_atStearoyl-coenzyme A desaturase 2 Scd2 124 1112 11 1415824_atHigh density lipoprotein

(HDL) binding proteinHdlbp 41 250 8 1449615_s_at

Hydroxysteroid (17-beta)dehydrogenase 7

Hsd17b7 15 97 7 1417871_at

Low density lipoprotein receptor Ldlr 39 340 7 1450383_atLow density lipoprotein

receptor-related protein 6Lrp6 27 206 7 1451022_at

Glycerol-3-phosphate acyltransferase,mitochondrial

Gpam 57 355 6 1425834_a_at

Sphingosine phosphate lyase 1 Sgpl1 53 248 5 1415893_atATP-binding cassette, sub-family

F (GCN20), member 1Abcf1 54 293 4 1452236_at

Lysophosphatidylglycerolacyltransferase 1

Lpgat1 66 235 4 1424350_s_at

3-Phosphoinositide dependentprotein kinase-1

Pdpk1 93 332 4 1416501_at

Glycerophosphodiester phosphodiesterasedomain containing 1

Gdpd1 185 767 4 1424077_at

Ribosomal protein L18 Rpl18 5114 5939 No change 1450372_a_atRibosomal protein L26 Rpl26 10,826 121,929 No change 1436995_a_at

Table 4Total number of genes detected in the 6 groups of mice and the number of CS sensitive genes

Genotype Treatment Total number of gene detected CS sensitive genes

Total Repressed Induced

WT Air 16,277 – – –WT 3 days CS 15,828 1702 1211 491WT 10 days CS 15,850 1879 1148 731

ATTP-KO Air 16,568 – – –ATTP-KO 3 Days CS 16,230 562 430 132ATTP-KO 10 days CS 16,107 2256 2241 15

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Analysis of genome-wide responses of lungs to inhaled CS shows the complexityof transcriptional response of the lung genome to CS and its possible modulation bydietary factors. When inhaled, CS affects the expression of numerous lung genes thathave been classified into several functional clusters that include oxidative stress,

Fig. 3. Summary of possible pathways by which AT may modulate the lung transcriptome. Theexpressions of �2000 genes were affected that included members of nuclear receptor superfamily,Nrf2:Keap1 regulated genes, heat-shock proteins (HSPs), HDLs and LDLs. The figure also suggestspossible ‘‘mechanisms’’ by which chronic AT deficiency may affect gene expression. Metabolism is knownto generate reactive oxygen and nitrogen metabolites (ROM and RNM, respectively) through pathwaysincluding the electron transport chains in mitochondria, cytochrome P450s in endoplasmic reticulum, andNAD(P)H oxidase in the plasma membrane. ROM and RNM may react with various cellular lipids andgenerate lipid peroxides which in turn affect critical thiol groups in proteins. These reactive intermediatesmay affect gene transcription. Thus AT may modulate these pathways via its ability to ‘‘scavenge’’ reactiveintermediates. Alternatively, AT or its metabolites generated in situ, or in extra-pulmonary organs such asthe liver, may also affect the expression of lung transcriptomes.

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xenobiotic metabolism, host defense, inflammation and circadian rhythm (Gebelet al., 2006; Maunders et al., 2007; Meng et al., 2006; Ning et al., 2004). We haveutilized GeneChips to monitor the CS responses of lungs from WT and ATTP-KO mice with the aim to uncover actions of AT in lung tissues. Table 4 summarizes

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the total number of genes affected by CS exposure for either 3 or 10 days as describedin Methods. The data show that the assay screened the responses of �16,000 lunggenes. The response of lung transcriptome to CS appears to be sensitive to boththe period of exposure and the genotype of the mouse. The data in Table 4 also sug-gest that the total numbers of repressed and induced genes appears to be more sen-sitive to the period of CS-exposures in the AT-deficient lungs of ATTP-KO mice ascompared to those of the WT lungs.

3.3. Induction of AhR driven genes

In order to validate our experimental and analytical approach for the identifica-tion of AT sensitive genes in the presence or the absence of CS, we first searched forthe battery of phase I and phase II genes which have previously been shown to beinduced in lungs by exposure to CS (Gebel et al., 2006; Rangasamy et al., 2004; Vil-lard et al., 1994). Fig. 4 shows a ‘‘heat-map’’ for expression of some of the represen-tative members of this cluster of transcriptionally regulated genes. The data showthat the expression of most of the genes was low in air breathing mice but wasinduced by breathing CS. Induction of cyp1a1 and cyp1b1 genes by CS in the lungsof rodents (Gebel et al., 2004, 2006; Iba et al., 2006) and in human lung tissue (Kimet al., 2004) has previously been reported and Fig. 4 validates our GeneChip assayand CS-exposure protocols. The data are particularly noteworthy because, unlikethe previous studies (Gebel et al., 2004, 2006), the activation of phases I and IIresponses observed in our studies were achieved at a much lower concentration oftotal suspended particles (60 mg/m3 vs 300 mg/m3) and after shorter exposure peri-ods (3 days vs 14 days). In addition, our mice underwent whole body exposure in

Fig. 4. Expression of Phase I and Phase II response genes in the lungs of WT and ATTP-KO mice andtheir modulation by exposures to CS. Each column shows pooled data from 5 mice. Each row shows theexpression of the identified gene relative to that in the six treatment groups. Green shows low expression,intermediate expression is indicated in black and red shows high expression.

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contrast to nose-only exposure used in many previous studies. CS in the exposurechambers used in the present studies contained a mixture of mainstream and side-stream smoke (Lofroth and Lazaridis, 1986; Witschi et al., 2004).

GeneChip data suggest that CS-induced expression of cyp1b1 is higher in AT-deficient lungs of ATTP-KO mice as compared to that from the lungs of CS-breath-ing WT mice with much higher lung AT levels. This observation suggests that ATmay modulate the expression of a transcriptionally regulated gene important inthe metabolism of carcinogens (Shen et al., 1994; Shimada and Fujii-Kuriyama,2004) and in carcinogenesis (Buters et al., 1999; Chang et al., 2007; Roos and Bolt,2005). Previous studies have shown that CS inhalation increases activities of enzymesencoded by cyp1 genes (Gairola, 1987). Cyp1a1 and 1b1 induction decreases benzo-pyrene induced formation of protein and DNA adducts, metabolites, conjugates andunmetabolized benzo[a]pyrene (Sagredo et al., 2006) suggesting possible mechanismsby which they may provide protective functions against carcinogens. Cyp1a1 andcyp1b1 are induced in Clara cells by 2,3,7,8-tetrachlorodibenzo-p-dioxin, a highaffinity ligand for AhR (Chang et al., 2006). Examination of our GeneChip datashowed that Clara cell marker gene, CC10, was highly expressed in lung homoge-nates suggesting that the Clara cell specific transcriptome was represented in the lungtissue homogenate and that AT may be affecting the expression of cyp1b1 in Claracells of mouse lungs.

To further validate the induction of cyp1b1 and Nqo1 detected by the GeneChipassay, quantitative real-time (qRT) PCR methodology was used to quantify theexpression of cyp1b1 mRNA in RNA samples isolated from each mouse lung fromeach treatment group. The data are shown in Fig. 5. Lung AT deficiency inflicted inATTP-KO mice potentiated CS-induced expression of cyp1b1 and Nqo1 detected inthe lungs of AT-normal WT mice. QRTPCR data reinforce the GeneChip data andsupport our proposition that lung AT-deficiency affects the expression of these genes.

Fig. 5. AT deficiency potentiates CS-induced expression of cyp1b1 and Nqo1 mRNAs. qRTPCR datafrom 6 groups of 5 months old female mice. The expression of each gene was normalized to the expressionof GAPDH in each sample. The fold-induction of each gene in the CS-exposed lungs was calculated on thebasis of the normalized expression of each gene in air breathing mice (fold change = 1). The error barsshow SEM. The data show that CS exposure induced cyp1b1 and Nqo1 expression that appears to beproportional to the time of exposure. The data also show that the induction of both the genes wassignificantly higher in the lungs of ATTP-KO mice deficient in AT.

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The expression of cyp1b1 and cyp1a1 in liver is transcriptionally regulated (Murrayet al., 2001; Tsuchiya et al., 2003) and is dependant on the presence of the AhR gene(Tijet et al., 2006). Data from in vivo studies in mice with deleted AhR gene suggestthat the expression of Nqo1 is also regulated by AhR (Tijet et al., 2006) in additionto another redox sensitive transcription factor, Nrf2 (Jaiswal, 2000; Rangasamyet al., 2004). Our GeneChip data also detected dysregulated expression of aromatichydrocarbon receptor translocator like (Arntl) mRNA. The significance of thisobservation is that lung AT appears to affect the expression of a gene which belongsto a TF-family that has conserved basic helix–loop–helix and PAS (Per, Arnt, Sim)domains and includes TFs such as AhR and Arnt (Wolting and McGlade, 1998) thatregulate cell’s responses to xenobiotics. Therefore, we evaluated the expressions ofAhR and Arntl by qRTPCR in the lungs of mice from the various treatment groups.The data, shown in Fig. 6, confirm the GeneChip data and provide quantitative andstatistical support for the modulation of Arntl expression by AT and by CS expo-sure. The data show that the expressions of Arntl and AhR were significantly lowerin the lungs of ATTP-KO as compared to those in the lungs of WT mice in theabsence of CS-challenge. However, when challenged with CS, the expression of Arntlwas significantly higher in the lungs of ATTP-KO mice as compared to their WTcontrols. This was seen in the lungs of mice exposed to CS for 3 or 10 days. AhRand Arnt regulate transcription of genes important in xenobiotic metabolism (Tijetet al., 2006). Since at least one of the molecular components of this pathway, i.e.

Fig. 6. AT-deficiency modulates the expression of Arntl and AhR genes in the lungs. The expression ofeach gene was normalized to the expression of GAPDH in each sample. The fold-induction of each gene inthe CS-exposed lungs was calculated on the basis of the normalized expression of each gene in airbreathing mice (fold change = 1). The error bars indicate SEM. The data suggest that CS exposurerepressed the expression of Arntl in the lungs of WT mice that have normal concentrations of AT. Incontrast, CS exposure induced the expression of Arntl gene in the lungs of ATTP-KO mice that aredeficient in AT. In the absence of CS challenge the expression of Arntl and AhR was lower in the lungs ofATTP-KO mice compared to those in the lungs of the WT mice. CS-induced induction of the AhR genewas very similar in the WT and ATTP-KO mice.

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Arntl appears to be regulated by lung AT in the absence of CS, our data suggest thatAT may play a role in transcriptional regulation of some of the AhR driven genes byendogenous metabolites such as those produced by lipid peroxidation. Arntl is also amember of the transcription factor network that regulates circadian rhythm (Takah-ata et al., 2000). A recent study has documented modulation of circadian rhythmgenes by CS in rat lungs (Gebel et al., 2006) and which may validate the present datafrom mouse lungs exposed to CS.

3.4. Modulation of Nrf2 driven genes

Does AT affect Nrf2 driven genes? As noted above, most of the current literaturepostulates that Nrf2 is a redox sensitive transcription factor that regulates theexpression of a battery of genes important in xenobiotic metabolism (Kensleret al., 2006; Rangasamy et al., 2004) and alleged to protect lung tissues from oxida-tive stress (Cho et al., 2006). However, Nrf2 may also be activated by mechanismsindependent of oxidative stress (Ho et al., 2005). If AT has antioxidant activityin vivo, then it is reasonable to hypothesize that AT-deficiency should augmentNrf2 activity displayed by induction of the specific Nrf2 driven genes. Genome-widesearch of AT sensitive lung genes identified a number of genes that are potentiallydriven by Nrf2. These genes included those encoding heme-oxygenase 1 (Hmox1)and catalytic and regulatory subunits of glutamate-cysteine ligase (Table 1 andFig. 4). The induction of these genes in the AT-deficient lungs of mice in the absenceof oxidant challenge of CS may be an indication of altered redox status in lungs ofATTP-KO mice because these genes are known to be induced by an increasedGSSG/GSH ratio (Bauer et al., 1998; Buzaleh and Batlle, 2005; Srisook and Cha,2005) and transition metal ions such as Fe3+ and Cd2+ (Alam et al., 1989; Linet al., 1990). We further addressed this indication from GeneChip data by quantifi-cation of the three Nrf2 driven mRNAs in the lungs of mice in our study. The data inFig. 7 show differential effects of AT-deficiency and CS on the expression of the threeNrf2 driven genes. The expression of Hmox1 was not significantly higher in the lungsof ATTP-KO when compared with its expression in air breathing lungs. CS breath-ing transiently induced Hmox1, and the induction was unaffected by AT deficiency.The induction of Gclm appears to be similar to that of Hmox1, suggesting that thesetwo Nrf2 driven genes are similarly regulated. However, in the lungs of air breathingmice, the expression of Gclc which encodes the catalytic subunit of the rate limitingenzyme in GSH synthesis was significantly higher in mice with AT-deficiency as com-pared to those with normal AT suggesting that Gclc expression is more sensitive toAT than the expression of its modulatory counterpart. CS activated Gclc expressionin the lungs of both WT and ATTP-KO. There was no significant effect of AT defi-ciency on CS-induced expression of Gclc. Collectively, the simplest interpretation ofthese data for the three well characterized Nrf2 driven genes is that AT and its pos-tulated antioxidant properties do not affect the actions of CS-oxidants in vivo on thiscluster of genes. The lack of effect of AT on CS-induced expression of Hmox1, Gclcand Gclm is in contrast to that noted above for the expression of cyp1b1 and Nqo1whose CS-induced expression was potentiated in the lungs of AT-deficient mice

Fig. 7. QRTPCR data for the expression of heme oxygenase 1 (Hmox1), glutamate-cysteine ligasecatalytic subunit (Gclc) and glutamate-cysteine ligase modulatory subunit (Gclm) mRNAs in the lungs ofWT and ATTP-KO mice breathing either air or CS. The data show differential effects of AT deficiency andCS on the expression of the three Nrf2 driven genes. The expression of each gene was normalized to theexpression of GAPDH in each sample. The fold-induction of each gene in the lungs from each group wascalculated on the basis of the normalized expression of each gene in air breathing mice (fold change = 1).The error bars indicate SEM. The expression of Hmox1 was not significantly different in the lungs ofATTP-KO when compared with its expression in air breathing lungs. CS breathing for 3 days inducedHmox1 and the induction was unaffected by AT deficiency. CS induced activation of Hmox1 appears to betransient. The CS-induced increase of Gclm appears to be similar to that of Hmox1. In the lungs of airbreathing mice the expression of Gclc was significantly higher in mice with AT-deficiency compared tothose with normal AT. CS activated Gclc expression in the lungs of both WT and ATTP-KO mice. Therewas no significant effect of AT deficiency on CS induced expression of Gclc.

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(Fig. 5). These observations prompt the speculation that AT, a lipophilic vitamin,affects the expression of genes encoding enzymes that are compartmentalized in lipo-philic environments; cyp1b1 and Nqo1 genes encode electron transport chains thatare sequestered in membranes whereas Hmox1, Gclc and Gclm genes encode pro-teins that are in the cytosolic compartments. The mechanisms by which AT mayselectively modulate such transcriptional responses remain to be furthercharacterized.

3.5. Modulation of immune-inflammatory genes

The largest cluster of functionally related genes that were modulated by AT andCS encode immune and inflammation related genes (Fig. 8). Respiratory tract har-bors lymphoid tissue which is primarily associated with the bronchus (Bienenstockand McDermott, 2005). The lung tissue used for GeneChip analysis was dissected

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free of large airways that presumably contain a portion of lung associated lymphoidtissues. The data in Fig. 8 suggest the presence of a large number of immune-inflam-mation related genes in lung tissue homogenates; these may be associated with thesmaller airways, alveolar parenchyma and residual pulmonary interstitium and cir-culation. AT deficiency altered the expression of this battery of genes in the lungsof both air breathing and CS breathing mice (Fig. 8). In air breathing mice, genesin clusters 1 and 2, and some genes in cluster 5, appear to be repressed. Most ofthe genes in clusters 1 and 2 code for immunoglobulins which are primarily expressedby B-cells. These data suggest that severe and chronic AT-deficiency of the ATTP-KO mice affects lung B-cell functions. These data from GeneChip analysis are sup-ported by qRTPCR data for the selected genes from these clusters (Fig. 9). The datashow that, in air breathing mice, AT-deficiency represses the expression of bothimmunoglobulin J-regions (IgJ) and immunoglobulin H-chains (IgH) and that oflymphotoxin-b (Ltbx), a transcriptionally regulated, NF-jB driven gene (Messer

Fig. 8. Expression of immune-inflammatory genes in the lungs of WT and ATTP-KO mice and theirmodulation by exposures to CS. Each column shows pooled data from 5 mice. Each row shows the relativeexpression of the identified gene in the six groups of mice. Blue indicates low expression, white indicatesintermediate expression and red indicates high expression. The affected genes were classified into sevenclusters based on the assigned functions of the encoded proteins. The expression of most of the genes inclusters 1–5 appears to be down-regulated in the lungs of air breathing ATTP-KO. Most of the genes inclusters 2–6 were down-regulated in the lungs of mice breathing CS suggesting immunosuppressive effectof CS. The expression of genes in cluster 7 was higher in the lungs of ATTP-KO mice suggesting arepressive role of lung AT in the expression of these genes.

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et al., 1990; Paul et al., 1990). A previous study has suggested that AT stimulatesproliferation of splenic lymphocytes, in vitro (Roy et al., 1991). AT deficiency in ratshas been shown to alter ultrastructure of mitochondria in reticulocytes and periph-eral lymphocytes (Lehmann and McGill, 1982). AT has also been suggested to pro-tect lymphoid cell lines when challenged by oxidized lipoproteins (Negre-Salvayreet al., 1991). In broilers, AT was shown to affect the proportion of CD4 T cells(Erf et al., 1998). A more recent study in piglets suggests that AT supplementationsignificantly increased IgA+ B-lymphocytes in Peyer’s patch, a gut associated lym-phoid tissue (Fragou et al., 2006), and in old mice AT appears to modulate Th1/Th2 balance through transcriptional mechanisms (Han et al., 2006). Hence, our data

Fig. 9. Modulatory role of AT and CS on the expression of lymphotoxin-b (Ltxb), heavy chain ofimmunoglobulin (IgH) and j-chain of immunoglobulin (IgJ) in the lungs of air breathing mice. The dataare from qRTPCR analysis. The expression of each gene was normalized to the expression of GAPDH ineach sample. The fold-induction of each gene in the lungs from each group was calculated on the basis ofthe normalized expression of each gene in air breathing mice (fold change = 1). The error bars indicateSEM. In air breathing mice the expression of each gene was significantly lower (P < 0.01) in the lungs fromATTP-KO mice as compared to that from WT mice. In the lungs of CS breathing ATTP-KO mice theexpression of each gene was significantly higher (p < 0.05-0.005) than that in the lungs of CS breathing WTmice after 10 days. In contrast, CS exposures repressed the expressions of these genes in AT normal lungsof WT mice. The arrow with * shows significant repression of IgH and IgJ genes in CS exposed lungs ofWT mice when compared with that of air breathing WT mice.

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from air breathing, 5-month-old female mice suggest that AT is required for B-cellfunctions and homeostasis in the lungs.

Data in Figs. 8 and 9 strongly suggest a role for lung AT in modulating immune-inflammation related mRNA expression. Biosynthesis of immunoglobulin heavychains recruits both transcriptional and DNA-recombination mechanisms (Harri-man et al., 1993; Kinoshita and Honjo, 2000) and signaling pathways that dependon members of protein kinase C (PKC) family and on NF-jB in B-cells (Moscatet al., 2003). Previous studies have suggested PKC to be target of AT (Azzi et al.,1999) possibly by modulation of a phosphatase activity (Chan et al., 2001). We spec-ulate that AT-sensitive immune-inflammatory related genes detected by the Gene-Chip assay and confirmed by qRTPCR analysis in RNA samples from mouselungs discussed here may be mediated through PKC, NF-jB pathways.

GeneChip data also suggest that the expression of immune-inflammatory genes isrepressed in the lungs of WT-mice breathing CS (Figs. 8 and 9). Previous studieshave documented similar effects of CS in lungs (Robbins et al., 2006; Robbinset al., 2005; Sopori et al., 1989), in T-cells (Chang et al., 1990) and in B-cells (vander Strate et al., 2006). A similar cluster of functionally related genes was repressedin the lungs of mice breathing ozone (Gohil et al., 2003a). In contrast, AT-deficiencyappears to induce the expression of some the same genes in the lungs of CS-breathingATTP-KO mice. These effects were most obvious for genes in cluster 1 (Fig. 8).These observations were reinforced by qRTPCR data for three of the genes(Fig. 9). CS-induced activation of Ltxb and IgH in the lungs of ATTP-KO micewas significantly higher (P < 0.05–0.005) than that in the lungs of CS breathingWT mice. These observations suggest that AT known to be present in lymphocytes(Kayden et al., 1984) is capable of modulating the expression of these genes. Themechanisms by which AT may affect the expression of these genes remain to becharacterized.

4. Concluding remarks

In this study, we have focused on the utility of genome-wide search for detectingthe diversity of the actions of AT in vivo. Our focus was on lung because this is avital organ that is subjected to considerable oxidative and non-oxidant stressorsfrom endogenous and environmental sources. The focus on mRNA was dictatedby the fact that it is an obligatory intermediate in the flow of molecular informationfrom the genome to the proteome and the availability of the methodology for anal-ysis of most of the mRNAs transcribed from the genome. The current knowledge ofmRNA metabolism suggests that the concentrations of mRNAs are primarily reg-ulated by interactions of transcription factors, RNA-polymerases and chromatin.Hence AT-sensitive changes in mRNAs must be caused, at least in part, by actionsof this vitamin or its metabolites either directly or indirectly through signaling cas-cades that converge on transcriptional machineries and their abilities to transcribethe genome. Comparative analyses of the lung transcriptomes (�16,000 mRNAs)from AT-normal and AT-deficient mice (obtained by the deletion of the ATTP

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gene) identified �2000 genes modulated by AT. Functional classification of theaffected genes identified diverse groups of genes that included members of nuclearreceptor superfamily and immune-inflammatory genes. Many of these gene-clusterswere differentially regulated in the lungs of mice breathing CS which is known tocontain free radicals and xenobiotics. Differential regulation of the lung transcrip-tome by CS in WT and ATTP-KO mice suggests significant role of AT in theexpression of lung genes. The precise mechanisms by which AT modulates theexpression of the lung genome and how such modulations may affect lung patho-biology needs further clarification.

Acknowledgements

Research was supported, in part by Philip Morris USA Inc. and Philip MorrisInternational, NIH ES 011985-02, ES 011985, USDA (2003-00915) and ClinicalNutrition Research Unit Pilot Research grant from University of California.

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