/SSTϩ neurons. Dbx1 mutant mice do not express any spontaneous respiratory behaviors in vivo. Moreover, they do not generate rhythmic inspiratory activity in isolated en bloc preparations even after acidic or serotonergic stimulation. These data indicate that preBötC core neurons represent a subset of a larger, more heterogeneous population of VLM Dbx1-derived neurons. These data indicate that Dbx1-derived neurons are essential for the expression and, we hypothesize, are responsible for the generation of respiratory behavior both in vitro and in vivo.

Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) is the primary transcription factor protecting cells from oxidative stress by regulating cytoprotective genes, including the antioxidant glutathione (GSH) pathway. GSH maintains cellular redox status and affects redox signaling, cell proliferation, and death. GSH homeostasis is regulated by de novo synthesis as well as GSH redox state; previous studies have demonstrated that Nrf2 regulates GSH homeostasis by affecting de novo synthesis. We report that Nrf2 modulates the GSH redox state by regulating glutathione reductase (GSR). In response to oxidants, lungs and embryonic fibroblasts (MEFs) from Nrf2-deficient (Nrf2−/−) mice showed lower levels of GSR mRNA, protein, and enzyme activity relative to wild type (Nrf2+/+). Nrf2−/− MEFs exhibited greater accumulation of glutathione disulfide and cytotoxicity compared to Nrf2+/+ MEFs in response to t-butylhydroquinone, which was rescued by restoring GSR. Microinjection of glutathione disulfide induced greater apoptosis in Nrf2−/− MEFs compared to Nrf2+/+ MEFs. In silico promoter analysis of the GSR gene revealed three putative antioxidant-response elements (ARE1, −44; ARE2, −813; ARE3, −1041). Reporter analysis, site-directed mutagenesis, and chromatin immunoprecipitation assays demonstrated binding of Nrf2 to two AREs distal to the transcription start site. Overall, Nrf2 is critical for maintaining the GSH redox state via transcriptional regulation of GSR and protecting cells against oxidative stress.

We have used a simple and efficient method to identify condition-specific transcriptional regulatory sites in vivo to help elucidate the molecular basis of sex-related differences in transcription, which are widespread in mammalian tissues and affect normal physiology, drug response, inflammation, and disease. To systematically uncover transcriptional regulators responsible for these differences, we used DNase hypersensitivity analysis coupled with high-throughput sequencing to produce condition-specific maps of regulatory sites in male and female mouse livers and in livers of male mice feminized by continuous infusion of growth hormone (GH). We identified 71,264 hypersensitive sites, with 1,284 showing robust sex-related differences. Continuous GH infusion suppressed the vast majority of male-specific sites and induced a subset of female-specific sites in male livers. We also identified broad genomic regions (up to ϳ100 kb) showing sex-dependent hypersensitivity and similar patterns of GH responses. We found a strong association of sex-specific sites with sex-specific transcription; however, a majority of sex-specific sites were >100 kb from sex-specific genes. By analyzing sequence motifs within regulatory regions, we identified two known regulators of liver sexual dimorphism and several new candidates for further investigation. This approach can readily be applied to mapping condition-specific regulatory sites in mammalian tissues under a wide variety of physiological conditions. Sexual dimorphism in gene expression is common in mammalian somatic tissues (23) and has broad implications for human health. Sex differences in gene expression may contribute to differences between men and women in the prevalence, extent, and progression of disease, including autoimmune diseases (54), kidney disease (37), cardiovascular disease (45), and liver diseases, such as hepatocellular carcinoma (9, 58). In addition, sex-related differences in pharmacokinetics and pharmacodynamics are common and may affect drug response (52). Sex-related differences in gene expression have been widely studied in liver, where they affect Ͼ1,000 transcripts (5, 51, 57) and impact physiological and pathophysiological functions ranging from lipid and fatty acid metabolism to xenobiotic metabolism and disease susceptibility (52). In the liver, sexrelated differences in gene expression are primarily determined by growth hormone (GH) signaling (3, 21), which shows important sex-related differences that reflect the sex-related differences in plasma GH profiles seen in many species, including rats, mice, and humans (53).The underlying mechanisms of sexual dimorphism in mammalian tissues have been only partly elucidated at the molecular level. In the male rat liver, intermittent plasma GH pulses repeatedly activate the latent cytoplasmic transcription factor STAT5b, whose activity is essential for sex-related differences in the liver (5). The more continuous, female-like pattern of pituitary GH secretion can be mimicked by continuous GH infusion in mal...

Oxidative stress results in protein oxidation and is involved in the pathogenesis of lung diseases such as chronic obstructive pulmonary disorder (COPD). Sulfiredoxin-1 (Srx1) catalyzes reduction of cysteine sulfinic acid to sulfenic acid in oxidized proteins and protects them from inactivation. This study examined the mechanism of transcriptional regulation of Srx1 and its possible protective role during oxidative stress associated with COPD. Nrf2, a transcription factor known to influence susceptibility to pulmonary diseases, upregulates Srx1 expression during oxidative stress caused by cigarette smoke exposure in the lungs of mice. Disruption of Nrf2 signaling by genetic knockout in mice or RNAi in cells downregulated the expression of Srx1. In silico analysis of the 5′-promoter flanking region of Srx1 identified multiple antioxidant response elements that are highly conserved. Reporter and chromatin-immunoprecipation assays demonstrated that ARE1 at −228 is critical for the Nrf2-mediated response. Attenuation of Srx1 expression with RNAi potentiated the toxicity of hydrogen peroxide (H 2 O 2 ), whereas overexpression of Srx1 protected against H 2 O 2 mediated cell death in vitro. Immunoblot analysis revealed dramatic decreases in Srx1 expression in lungs from patients with COPD relative to non-emphysematous lungs together with a decline in Nrf2 protein.Thus, Srx1, a key Nrf2-regulated gene, contributes to protection against oxidative injury in the lung. †Address correspondence to:

Exogenously administered endothelin (ET) elicits both pressor and depressor responses through the ETA and/or the ETB receptor on vascular smooth muscle cells and ETB on endothelial cells. To test whether ETB has pressor or depressor effects under basal physiological conditions, we determined arterial blood pressure (BP) in ETB-deficient mice obtained by crossing inbred mice heterozygous for targeted disruption of the ETB gene with mice homozygous for the piebald ( s) mutation of the ETB gene ( ET B s/s ). F1 ET B −/s and ET B +/s progeny share an identical genetic background but have ETB levels that are ∼[Formula: see text]and [Formula: see text], respectively, of wild-type mice ( ET B +/+ ). BP in ET B −/s mice was significantly higher, by ∼20 mmHg, than that in ET B +/s or ET B +/+ mice. Immunoreactive ET-1 concentration in plasma as well as respiratory parameters was not different between ET B −/s and ET B +/s mice. A selective ETB antagonist, BQ-788, increased BP in ET B +/s and ET B +/+ but not in ET B −/s mice. Pretreatment with indomethacin, but not with N G-monomethyl-l-arginine, can attenuate the observed pressor response to BQ-788. The selective ETA antagonist BQ-123 did not ameliorate the increased BP in ET B −/s mice. Moreover, BP in mice heterozygous for targeted disruption of the ETA gene was not different from that in wild-type controls. These results suggest that endogenous ET elicits a depressor effect through ETB under basal conditions, in part through tonic production of prostaglandins, and not through secondary mechanisms involving respiratory control or clearance of circulating ET.

ABSTRACT:CYP2A13, CYP2B6, and CYP2F1, which are encoded by neighboring cytochrome P450 genes on human chromosome 19, are active in the metabolic activation of many drugs, respiratory toxicants, and chemical carcinogens. To facilitate studies on the regulation and function of these human genes, we have generated a CYP2A13/2B6/2F1-transgenic (TG) mouse model (all *1 alleles). Homozygous transgenic mice are normal with respect to gross morphological features, development, and fertility. The tissue distribution of transgenic mRNA expression agreed well with the known respiratory tract-selective expression of CYP2A13 and CYP2F1 and hepatic expression of CYP2B6 in humans. CYP2A13 protein was detected through immunoblot analyses in the nasal mucosa (NM) (ϳ100 pmol/mg of microsomal protein; similar to the level of mouse CYP2A5) and the lung (ϳ0.2 pmol/mg of microsomal protein) but not in the liver of the TG mice. CYP2F1 protein, which could not be separated from mouse CYP2F2 in immunoblot analyses, was readily detected in the NM and lung but not the liver of TG/Cyp2f2-null mice, at levels 10-and 40-fold, respectively, lower than that of mouse CYP2F2 in the TG mice. CYP2B6 protein was detected in the liver (ϳ0.2 pmol/mg of microsomal protein) but not the NM or lung (with a detection limit of 0.04 pmol/mg of microsomal protein) of the TG mice. At least one transgenic protein (CYP2A13) seems to be active, because the NM of the TG mice had greater in vitro and in vivo activities in bioactivation of a CYP2A13 substrate, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (a lung carcinogen), than did the NM of wild-type mice.

No abstract

We studied respiratory functions in mutant mice deficient in endothelin-1 (ET-1) generated by gene targeting. In conscious adult mice heterozygous for ET-1 gene mutation (ET+/- heterozygous mice), arterial PO2 was significantly lower, PCO2 tended to be higher, and pH tended to be lower than in wild-type littermates. When these conscious mice breathed room air, respiratory minute volume and rate, determined by body plethysmography, were not significantly different between the two groups. However, when ET+/- heterozygous mice were subjected to systemic hypoxia (1:1 air-N2) or hypercapnia (5% CO2-95% O2), increases in respiratory minute volume were significantly attenuated. In conscious newborn ET-/- homozygous mice delivered by cesarean section and tracheotomized, ventilatory responses to systemic hypoxia and hypercapnia, regularly observed in newborn wild-type mice, were almost totally absent. In urethan-anesthetized adult ET+/- heterozygous mice, increases in phrenic nerve discharges in response to hypoxia and hypercapnia were significantly attenuated. Our results demonstrate that ventilatory responses to hypoxia and hypercapnia are impaired in ET-1-deficient mice and suggest that endogenous ET-1 participates in the physiological control of ventilation.