Differential effects of ozone on airway and tissue mechanics in obese mice.

Obesity is an important risk factor for asthma. We recently reported increased ozone (O(3))-induced hyperresponsiveness to methacholine in obese mice (Shore SA, Rivera-Sanchez YM, Schwartzman IN, and Johnston RA. J Appl Physiol 95: 938-945, 2003). The purpose of this study was to determine whether this increased hyperresponsiveness is the result of changes in the airways, the lung tissue, or both. To that end, we examined the effect of O(3) (2 parts/million for 3 h) on methacholine-induced changes in lung mechanics with the use of a forced oscillation technique in wild-type C57BL/6J mice and mice obese because of a genetic deficiency in leptin (ob/ob mice). In ob/ob mice, O(3) increased baseline values for all parameters measured in the study: airway resistance (Raw), lung tissue resistance (Rtis), lung tissue damping (G) and elastance (H), and lung hysteresivity (eta). In contrast, no effect of O(3) on baseline mechanics was observed in wild-type mice. O(3) exposure significantly increased Raw, Rtis, lung resistance (Rl), G, H, and eta responses to methacholine in both groups of mice. For G, Rtis, and Rl there was a significant effect of obesity on the response to O(3). Our results demonstrate that both airways and lung tissue contribute to the hyperresponsiveness that occurs after O(3) exposure in wild-type mice. Our results also demonstrate that changes in the lung tissue rather than the airways account for the amplification of O(3)-induced hyperresponsiveness observed in obese mice.

DNA sequence variants of the platelet-derived growth factor A-chain gene.

BACKGROUND: Platelet-derived growth factor A-chain (PDGF-A) is a potent connective tissue mitogen implicated in lung growth and development. PDGF-A may have a role in asthma through effects on fibroblasts and bronchial smooth muscle cells. OBJECTIVE: To test the hypothesis that there exist variations in the PDGF-A gene associated with the asthma phenotype. METHODS: We screened genomic DNA from normal and asthmatic subjects using single-stranded conformational polymorphism (SSCP) for mutations in the promoter and all seven exons of the gene. RESULTS: Four transition polymorphisms (three novel) were identified: one each in exons 3 and 4 (overall population allele frequencies 0.18 and 0.02, respectively) which did not alter the protein sequence, one in exon 4 (frequency 0.005) which resulted in a valine to isoleucine substitution, and one in intron 5 (frequency 0.5). The intron 5-sequence variant is close to the 3' end of exon 5 but does not appear to affect alternative splicing of PDGF-A exon 6 RNA. The frequencies of the polymorphisms in exons 3 and intron 5 did not differ between the asthmatic and non-asthmatic subjects, but there was a significant frequency difference between Caucasian and African-American subjects for each of these polymorphisms (P = 0.03 and 0.003, respectively). CONCLUSION: No association was found between the sequence variants in the PDGF-A gene and the development of asthma. However, the allele frequency of some of the sequence variants differed between the Caucasian and African-American subjects.

The transcription factor early growth-response factor 1 modulates tumor necrosis factor-alpha, immunoglobulin E, and airway responsiveness in mice.

Early growth-response factor 1 (Egr-1) is a sequence-specific transcription factor that plays a regulatory role in the expression of many genes important in inflammation, cell growth, apoptosis, and the pathogenesis of disease. In vitro studies suggest that Egr-1 is capable of regulating the expression of tumor necrosis factor-alpha (TNF-alpha) and other genes involved in airway inflammation and reactivity following allergen stimulation. On the basis of these data, we hypothesized that in the absence of Egr-1, the TNF-alpha response and subsequent downstream inflammatory events that usually follow allergen challenge would be diminished. To test our hypothesis Egr-1 knock-out (KO) mice were examined in an ovalbumin (OVA)-induced model of airway inflammation and reactivity, and compared with identically treated wild-type (WT) control mice. In response to OVA sensitization and airway challenge, KO mice had diminished TNF-alpha mRNA and protein in the lungs and mast cells compared with WT mice. Interestingly, the KO mice had elevated IgE levels at baseline and after allergen challenge compared with WT mice. Furthermore, the airways of KO mice were hyporesponsive to methacholine challenge at baseline and after allergen challenge. These data indicate that Egr-1 modulates TNF-alpha, IgE, and airway responsiveness in mice.

Polymorphism of the beta(2)-adrenergic receptor gene and desensitization in human airway smooth muscle.

We examined the influence of two common polymorphic forms of the beta(2)-adrenergic receptor (beta(2)AR): the Gly16 and Glu27 alleles, on acute and long-term beta(2)AR desensitization in human airway smooth muscle (HASM) cells. In cells from 15 individuals, considered without respect to genotype, pretreatment with Isoproterenol (ISO) at 10(-7) M for 1 h or 24 h caused approximately 25% and 64% decreases in the ability of subsequent ISO (10(-6) M) stimulation to reduce HASM cell stiffness as measured by magnetic twisting cytometry. Similar results were obtained with ISO-induced cyclic adenosine monophosphate (cAMP) as the outcome indicator. Data were then stratified post hoc by genotype. Cells containing at least one Glu27 allele (equivalent to presence of the Gly16Glu27 haplotype) showed significantly greater acute desensitization than did cells with no Glu27 allele, whether ISO-induced cell stiffness (34% versus 19%, p < 0.03) or cAMP formation (58% versus 11%, p < 0.02) was measured. Likewise, cells with any Glu27 allele showed greater long-term desensitization of cell stiffness and cAMP formation responses than did cells without the Glu27 allele. The distribution of genotypes limited direct conclusions about the influence of the Gly16 allele. However, presence of the Gly16Gln27 haplotype was associated with less acute and long-term desensitization of ISO-induced cAMP formation than was seen in cells without the Gly16Gln27 haplotype (14% versus 47%, p < 0.09 for short-term desensitization; 32% versus 84%, p < 0.01 for long-term desensitization), suggesting that the influence of Glu27 is not through its association with Gly16. The Glu27 allele was in strong linkage disequilibrium with the Arg19 allele, a polymorphic form of the beta(2)AR upstream peptide of the 5'-leader cistron of the beta(2)AR, and this polymorphism in the beta(2)AR 5'-flanking region may explain the effects of the Glu27 allele. Cells with any Arg19 allele showed significantly greater acute and long-term desensitization of ISO-induced cAMP formation than did cells without the Arg19 allele (54% versus 2%, p < 0.01 for short-term desensitization; 73% versus 35%, p < 0.05 for long-term desensitization). Similar results were obtained for ISO-induced changes in cell stiffness. Thus, the presence of the Glu27 allele is associated with increased acute and long-term desensitization in HASM.

IL-1beta induces eotaxin gene transcription in A549 airway epithelial cells through NF-kappaB.

Eotaxin is an asthma-related C-C chemokine that is produced in response to interleukin-1beta (IL-1beta). We detected an increase in newly transcribed eotaxin mRNA in IL-1beta-stimulated airway epithelial cells. Transient transfection assays using promoter-reporter constructs identified a region as essential for IL-1beta-induced increases in eotaxin transcription. Using site-directed mutagenesis, we found that a nuclear factor-kappaB (NF-kappaB) site located 46 bp upstream from the transcriptional start site was both necessary and sufficient for IL-1beta induction of reporter construct activity. Electrophoretic mobility shift assay demonstrated that IL-1beta-stimulated airway epithelial cells produced p50 and p65 protein that bound this site in a sequence-specific manner. The functional importance of the NF-kappaB site was demonstrated by coexpression experiments in which increasing doses of p65 expression vector were directly associated with reporter activity exclusively in constructs with an intact NF-kappaB site (r(2) = 0.97, P = 0.002). Moreover, IL-1beta-induced increases in eotaxin mRNA expression are inhibited by inhibitors of NF-kappaB. Our findings implicate NF-kappaB and its binding sequence in IL-1beta-induced transcriptional activation of the eotaxin gene.

High-level expression of Egr-1 and Egr-1-inducible genes in mouse and human atherosclerosis.

To understand the mRNA transcript profile in the human atherosclerotic lesion, RNA was prepared from the fibrous cap versus adjacent media of 13 patients undergoing carotid endarterectomy. cDNA expression arrays bearing 588 known genes indicated that lesions express unexpectedly high levels of the early growth response gene, Egr-1 (NGFI-A), a zinc-finger transcription factor that modulates a cluster of stress-responsive genes including PDGF and TGF-beta. Expression of Egr-1 was an average of 5-fold higher in the lesion than in the adjacent media, a result confirmed by RT-PCR, and many Egr-1-inducible genes were also strongly elevated in the lesion. Time-course analyses revealed that Egr-1 was not induced ex vivo. Immunocytochemistry indicated that Egr-1 was expressed prominently in the smooth muscle-actin positive cells, particularly in areas of macrophage infiltration, and in other cell types, including endothelial cells. Induction of atherosclerosis in LDL receptor-null mice by feeding them a high-fat diet resulted in a progressive increase in Egr-1 expression in the aorta. Thus, induction of Egr-1 by atherogenic factors may be a key step in coordinating the cellular events that result in vascular lesions.

The biology of 5-lipoxygenase: function, structure, and regulatory mechanisms.

5-Lipoxygenase (5-LO) catalyzes the two-step conversion of arachidonic acid to leukotriene A4 (LTA4). The first step consists of the oxidation of arachidonic acid to the unstable intermediate 5-hydroperoxyeicosatetraenoic acid (5-HPETE), and the second step is the dehydration of 5-HPETE to form LTA4. These events are the first committed reactions leading to the synthesis of all leukotrienes and play a critical role in controlling leukotriene production. 5-LO has evolved many complex structural features and regulatory mechanisms to allow it to fulfill this highly specialized role. The biology of 5-LO is reviewed here with an emphasis on enzymatic function, protein and gene structure, essential cofactors, and the many regulatory mechanisms controlling its expression.

Vascular smooth muscle cells express the transcriptional corepressor NAB2 in response to injury.

The early growth response 1 (Egr-1 or NGFI-A) gene product is a zinc finger protein transcription factor which has been implicated in the regulation of genes differentially expressed during the development of vascular disease. Egr-1 activity is regulated by alterations in the amount of protein, as well as protein-protein interactions with positive and negative transcriptional cofactors. NGFI-A-binding protein 2 (NAB2) is an example of a negative transcriptional cofactor capable of binding directly to Egr-1 and repressing Egr-1-mediated transcription. In this study, we show that NAB2 is rapidly and transiently expressed in vascular smooth muscle cells (VSMC) in response to the model agonist phorbol 12-myristate 13-acetate (PMA). This induction occurs at the protein as well as mRNA level, and the time course of induction trails closely behind that of Egr-1. NAB2 expression in VSMC is capable of inhibiting Egr-1 dependent gene expression in response to either PMA or fibroblastic growth factor-2 (FGF-2). In an in vivo model of mechanical arterial injury NAB2 levels also increase transiently in VSMC at a time when Egr-1 is elevated. It is possible that NAB2 is part of a negative-feedback mechanism which serves to down-regulate Egr-1-mediated gene transcription in injured VSMC.

Mutations in the human 5-lipoxygenase gene.

Our data demonstrate the presence of a naturally occurring family of alleles in the core promoter of the 5-LO gene, which is characterized by the deletion or addition of consensus Sp1 (-GGGCGG) and Egr-1 (-GCGGGGGCG-) binding motifs. Each of the variant alleles can bind Sp1 and Egr-1 protein, as indicated by EMSA and supershift analysis with nuclear extracts. In addition, preliminary data from CAT reporter assays indicate that these alleles are less effective than the wild-type allele in initiating 5-LO gene expression. Whether patients harboring the various alleles identified herein have different capacities to transcribe the 5-LO gene and the importance of such potential regulation to the clinical expression of 5-LO have yet to be determined.

Pharmacogenetic association between ALOX5 promoter genotype and the response to anti-asthma treatment.

Clinically similar asthma patients may develop airway obstruction by different mechanisms. Asthma treatments that specifically interfere with the 5-lipoxygenase (ALOX5) pathway provide a method to identify those patients in whom the products of the ALOX5 pathway (that is, the leukotrienes) contribute to the expression of the asthma phenotype. Failure of an asthma patient to respond to treatment with ALOX5-pathway modifiers indicates that leukotrienes are not critical to the expression of the asthmatic phenotype in that patient. We previously defined a family of DNA sequence variants in the core promoter of the gene ALOX5 (on chromosome 10q11.2) associated with diminished promoter-reporter activity in tissue culture. Because expression of ALOX5 is in part transcriptionally regulated, we reasoned that patients with these sequence variants may have diminished gene transcription, and therefore decreased ALOX5 product production and a diminished clinical response to treatment with a drug targeting this pathway. Such an effect indicates an interaction between gene promoter sequence variants and drug-treatment responses, that is, a pharmacogenetic effect of a promoter sequence on treatment responses.

Genetic determinants of 5-lipoxygenase transcription.

BACKGROUND: 5 Lipoxygenase (5-LO) is a critical enzyme in the production of the leukotrienes. We have identified a series of mutations in the 5-LO gene that modify gene transcription. These mutations consist of addition of an Sp-1 binding motif (-GGGCGG-) or deletion of one or two Sp-1 binding motifs in the 5-LO core promoter. METHODS: Mutant forms of the 5-LO core promoter were placed in a chloramphenicol acetyl transferase (CAT) reporter construct using either HeLa or SL-2 cells. RESULTS: In HeLa cells all of the mutant forms are less effective in driving CAT reporter activity than the wild-type promoter. In SL-2 cells the construct containing the addition mutation was more effective in driving CAT reporter activity, while the constructs containing the deletion mutations were less effective. CONCLUSIONS: These data indicate that naturally occurring mutations in the 5-LO core promoter modify gene transcription in vitro.

CREB-binding protein is a nuclear integrator of nuclear factor-kappaB and p53 signaling.

Transcriptional coactivators may function as nuclear integrators by coordinating diverse signaling events. Here we show that the p65 (RelA) component of nuclear factor-kappaB (NF-kappaB) and p53 mutually repress each other's ability to activate transcription. Additionally, tumor necrosis factor-activated NF-kappaB is inhibited by UV light-induced p53. Both p65 and p53 depend upon the coactivator CREB-binding protein (CBP) for maximal activity. Increased levels of the coactivator relieve p53-mediated repression of NF-kappaB activity and p65-mediated repression of p53-dependent gene expression. Nuclear competition for limiting amounts of CBP provides a novel mechanism for altering the balance between the expression of NF-kappaB-dependent proliferation or survival genes and p53-dependent genes involved in cell cycle arrest and apoptosis.

cAMP-response-element-binding-protein-binding protein (CBP) and p300 are transcriptional co-activators of early growth response factor-1 (Egr-1).

Egr-1 (early-growth response factor-1) is a sequence-specific transcription factor that plays a regulatory role in the expression of many genes important for cell growth, development and the pathogenesis of disease. The transcriptional co-activators CBP (cAMP-response-element-binding-protein-binding protein) and p300 interact with sequence-specific transcription factors as well as components of the basal transcription machinery to facilitate RNA polymerase II recruitment and transcriptional initiation. Here we demonstrate a unique way in which Egr-1 physically and functionally interacts with CBP/p300 to modulate gene transcription. CBP/p300 potentiated Egr-1 mediated expression of 5-lipoxygenase (5-LO) promoter-reporter constructs, and the degree of trans-activation was proportional to the number of Egr-1 consensus binding sites present in wild-type and naturally occurring mutants of the 5-LO promoter. The N- and C-terminal domains of CBP interact with the transcriptional activation domain of Egr-1, as demonstrated by a mammalian two-hybrid assay. Direct protein-protein interactions between CBP/p300 and Egr-1 were demonstrated by glutathione S-transferase fusion-protein binding and co-immunoprecipitation/Western-blot studies. These data suggest that CBP and p300 act as transcriptional co-activators for Egr-1-mediated gene expression and that variations between individuals in such co-activation could serve as a genetic basis for variability in gene expression.

Egr-1 and Sp1 interact functionally with the 5-lipoxygenase promoter and its naturally occurring mutants.

5-Lipoxygenase (5-LO), an enzyme essential for the formation of leukotrienes, is functionally modulated by a number of mechanisms, including transcriptional controls. The 5-LO promoter has a unique G+C-rich sequence, located between 176 and 147 base pairs upstream of the ATG translation start site, which contains five tandem Sp1 (a zinc-finger transcription factor) consensus binding sites overlapping five tandem early growth response protein 1 (Egr-1), a zinc-finger transcription factor, consensus binding sites. A family of naturally occurring mutations has been identified that consists of additions or deletions of these binding sites. The role of these overlapping Sp1/Egr-1 sites in the regulation of 5-LO transcription and the effects of these mutations on transcriptional regulatory mechanisms are unknown. We now show that Sp1 and Egr-1 bind specifically to the G+C-rich promoter sequence using in vitro deoxyribonuclease I footprinting. Both Sp1 and Egr-1 activate 5-LO promoter-reporter constructs in a minimally active drosophila SL2 cotransfection system, and the G+C-rich sequence is involved in this process. Moreover, studies comparing mutant promoter function indicate that both Sp1 and Egr-1 trans-activation are proportional to the number of Sp1/Egr-1 consensus binding sites within the G+C-rich sequence. It is possible that basal and inducible 5-LO gene transcriptions are mediated by an interplay of Sp1, Egr-1, and other transcription factors within the G+C-rich promoter region, and the naturally occurring mutations alter transcription by modifying their trans-activation potential.

Inducible PDGF A-chain transcription in smooth muscle cells is mediated by Egr-1 displacement of Sp1 and Sp3.

Platelet-derived growth factor (PDGF) A-chain is expressed by vascular smooth muscle cells (SMC) in a variety of pathological settings. Phorbol 12-myristate 13-acetate (PMA) increases A-chain transcription and was used as a model agonist. Transient transfection analysis identified a region in the promoter that is required for inducibility, located between base pairs -71 and -55 from the transcription start site. This region contains overlapping recognition elements for members of the Sp and Egr families. Egr-1 transcript and protein increased after PMA treatment, whereas Sp1 and Sp3 levels remain unchanged. Egr-1 expression and PDGF A-chain promoter activity also increased in cells exposed to PDGF or mechanical injury. In vitro binding assays demonstrated that Egr-1, Sp1, and Sp3 can bind to this promoter region and that increasing Egr-1 can displace both Sp1 and Sp3. In an in vivo model of arterial injury, Egr-1 expression was induced concurrently with the expression of PDGF-A in SMC. Displacement of Sp1 and Sp3 by Egr-1 is correlated with inducible PDGF A-chain expression in the vessel wall.

Naturally occurring mutations in the human 5-lipoxygenase gene promoter that modify transcription factor binding and reporter gene transcription.

Five lipoxygenase (5-LO) is the first committed enzyme in the metabolic pathway leading to the synthesis of the leukotrienes. We examined genomic DNA isolated from 25 normal subjects and 31 patients with asthma (6 of whom had aspirin-sensitive asthma) for mutations in the known transcription factor binding regions and the protein encoding region of the 5-LO gene. A family of mutations in the G + C-rich transcription factor binding region was identified consisting of the deletion of one, deletion of two, or addition of one zinc finger (Sp1/Egr-1) binding sites in the region 176 to 147 bp upstream from the ATG translation start site where there are normally 5 Sp1 binding motifs in tandem. Reporter gene activity directed by any of the mutant forms of the transcription factor binding region was significantly (P < 0.05) less effective than the activity driven by the wild type transcription factor binding region. Electrophoretic mobility shift assays (EMSAs) demonstrated the capacity of wild type and mutant transcription factor binding regions to bind nuclear extracts from human umbilical vein endothelial cells (HUVECs). These data are consistent with a family of mutations in the 5-LO gene that can modify reporter gene transcription possibly through differences in Sp1 and Egr-1 transactivation.

Na+/H+ exchange in pulmonary artery smooth muscle from spontaneously hypertensive and Wistar-Kyoto rats.

Na+/H+ exchanger regulation of intracellular pH (pHi) may play a key permissive role in the mitogen-induced vascular smooth muscle cell growth that occurs in systemic and pulmonary vascular remodeling. Spontaneously hypertensive rats (SHR) have increased Na+/H+ exchange in systemic vessels as well as greater systemic vascular remodeling compared with normotensive Wistar-Kyoto rats (WKY). In contrast to WKY, SHR demonstrate only mild pulmonary hypertension and no increased remodeling to hypoxia compared with WKY. We therefore wondered whether Na+/H+ exchange in pulmonary artery smooth muscle (PASM) of SHR might not be elevated compared with WKY. Baseline pHi, Vmax, pK0.5, and Hill coefficient were compared in 12- to 14-wk-old SHR and WKY PASM and aortic smooth muscle (AoSM) segments by ratio fluorescence spectroscopy. The Vmax, pK0.5, and Hill coefficient were significantly increased in SHR AoSM segments compared with WKY AoSM segments (53, 0.55, and 53%, respectively; P < 0.05). There were no differences in these values between SHR and WKY PASM segments, unlike the AoSM segments. We conclude that the Na+/H+ exchanger activity in PASM in the SHR is the same as in the WKY, which is in contrast to systemic arteries where Na+/H+ exchange is greater in the SHR.