Association of a missense mutation in the NOS3 gene with exhaled nitric oxide levels.

There is evidence that genetic factors affect nitric oxide formation and that sequence variants in the nitric oxide synthase genes contribute to the observed variance of nitric oxide levels in exhaled air (fraction of expired nitric oxide, FENO) in subjects with asthma. We identified a strong association between a known functional NOS3 missense sequence variant in the endothelial nitric oxide gene (G894T) and FENO level in a cohort of subjects with asthma. Age- and sex-adjusted FENO levels were lowest in asthmatic subjects with the TT genotype (geometric mean FENO [95% CI] = 7.17 [4.48 to 11.48] ppb) and were significantly higher in those with either the GT genotype (geometric mean FENO [95% CI] = 17.11 [13.80 to 21.23] ppb) or the GG genotype (geometric mean FENO [95% CI] = 12.06 [9.91 to 14.67] ppb) (F2,59 = 5.97, p = 0.004). The G894T DNA variant explained 16.3% of the residual variance in FENO levels. Our results demonstrate that the endothelial nitric oxide synthase, a nitric oxide synthase constitutively expressed in epithelial cells, plays an important role in determining measured levels of exhaled nitric oxide, a marker of the asthmatic condition

Endothelial nitric oxide synthase variants in cystic fibrosis lung disease.

Variants in the genes encoding for the nitric oxide synthases may act as disease modifier loci in cystic fibrosis, affecting both an individual\u27s nitric oxide level and pulmonary function. In this study, the 894G/T variant in exon 7 of the endothelial nitric oxide synthase gene was related to exhaled nitric oxide and pulmonary function in 70 cystic fibrosis patients who were aged 14.8 +/- 6.9 years (mean +/- SD), with a FEV1 of 69.4 +/- 24.8% predicted. Although there was no association between endothelial nitric oxide synthase genotypes and exhaled nitric oxide in males, nitric oxide levels were significantly higher in female cystic fibrosis patients with an 894T mutant allele, compared with female patients homozygous for the 894G wild-type allele (7.0 +/- 4.4 versus 3.6 +/- 1.9 parts per billion, p = 0.02). Furthermore, in female patients, colonization of airways with Pseudomonas aeruginosa was significantly (p \u3c 0.05) less frequent when carrying an 894T mutant allele as compared with wild type. These data suggest that the 894T variant in the endothelial nitric oxide synthase gene is associated with increased airway nitric oxide formation in female cystic fibrosis patients, possibly affecting colonization of airways with P. aeruginosa

Monocyte chemoattractant protein-4 core promoter genetic variants: influence on YY-1 affinity and plasma levels.

Monocyte chemoattractant protein-4 (MCP-4) is a CC chemokine implicated in the recruitment of eosinophils, monocytes, and T-lymphocytes in diseases of mucosal inflammation, including asthma. We tested the hypothesis that there is a genetic basis for differences in MCP-4 expression among individuals by evaluating the effects of core promoter variants on MCP-4 expression. We identified two single-nucleotide T-to-C polymorphisms in the MCP-4 core promoter that occur 896 and 887 base pairs preceding the transcription initiation site. The -887 variant alters a consensus binding motif for the transcription factor YY-1. Electrophoretic mobility shift assay demonstrated that YY-1 containing nuclear extracts from tumor necrosis factor-alpha-stimulated peripheral blood mononuclear cells had greater avidity for the wild-type (YY-1 motif intact) sequence than for the variant sequence. Increasing doses of a YY-1 expression vector induced significantly greater reporter activity from MCP-4 core promoter expression constructs of the wild-type compared with the variant sequence in transient transfection experiments. The external validity of these observations was demonstrated by measuring plasma levels of MCP-4 from individuals with the alternative forms of the gene. Individuals bearing haplotypic variants of the MCP-4 core promoter that avidly bind the transcription factor YY-1 had higher plasma levels of MCP-4 than did individuals with variants with lower binding avidity (490, 360, and 360 pg/ml; P \u3c 0.01). Our findings suggest that the MCP-4 core promoter YY-1 binding motif is functional, modulates the transcriptional regulation of the MCP-4 gene, and that part of the variance in the systemic expression of MCP-4 is determined by core promoter genetic variants

Cloning and functional analysis of the mouse 5-lipoxygenase promoter.

5-lipoxygenase (ALOX5), an enzyme essential for the formation of all leukotrienes, is highly regulated at multiple levels, including gene transcription. The human ALOX5 promoter sequence has been cloned and is well characterized. Several important cis-acting elements have been identified including a G+C-rich sequence approximately 145-179 base pairs (bp) upstream from the ATG start codon. This region contains consensus-binding sites for the transcription factor serum protein 1, a zinc-finger transcription factor (SP1) and early growth-response protein 1, a zinc-finger transcription factor (EGR-1) and is unique in that functionally significant polymorphisms alter these sequences. To further understand the significance of these polymorphisms and other regulatory sequences in the promoter we cloned approximately 2,000 bp of the mouse promoter sequence from a 129/SvJ BAC library for direct comparison with the human gene. Like the human promoter, the mouse Alox5 promoter lacks a TATA box and has multiple start sites. The first 292 bp immediately upstream of the translational start site function as a core promoter that is capable of mediating high basal transcription in RAW cells but not 3T3 cells. There are vast differences in the distribution of consensus cis elements between human and mouse genes; however, three areas of strong homology exist and they contain consensus-binding sites for the SP1, GATA, GGAGA, and ETS family of transcription factors. We show that Sp1/Sp3 is essential for constitutive promoter-reporter activity

IFN regulatory factor-1 regulates IFN-gamma-dependent cathepsin S expression.

Cathepsin S is a cysteine protease with potent endoproteolytic activity and a broad pH profile. Cathepsin S activity is essential for complete processing of the MHC class II-associated invariant chain within B cells and dendritic cells, and may also be important in extracellular matrix degradation in atherosclerosis and emphysema. Unique among cysteine proteases, cathepsin S activity is up-regulated by IFN-gamma. Given its importance, we sought to elucidate the pathway by which IFN-gamma increases cathepsin S expression. Our data demonstrate that the cathepsin S promoter contains an IFN-stimulated response element (ISRE) that is critical for IFN-gamma-induced gene transcription in a cell line derived from type II alveolar epithelial (A549) cells. IFN response factor (IRF)-2 derived from A549 nuclear extracts associates with the ISRE oligonucleotide in gel shift assays, but is quickly replaced by IRF-1 following stimulation with IFN-gamma. The time course of IRF-1/ISRE complex formation correlates with increased levels of IRF-1 protein and cathepsin S mRNA. Overexpression of IRF-1, but not IRF-2, markedly augments cathepsin S promoter activity in A549 cells. Furthermore, overexpression of IRF-1 increases endogenous cathepsin S mRNA levels in 293T epithelial cells. Finally, freshly isolated bone marrow cells from IRF-1(-/-) mice fail to up-regulate cathepsin S activity in response to IFN-gamma. Thus, IRF-1 is the critical transcriptional mediator of IFN-gamma-dependent cathepsin S activation. These data elucidate a new pathway by which IRF-1 may affect MHC class II processing and presentation