A Modified RMCE-Compatible Rosa26 Locus for the Expression of Transgenes from Exogenous Promoters

Generation of gain-of-function transgenic mice by targeting the Rosa26 locus has been established as an alternative to classical transgenic mice produced by pronuclear microinjection. However, targeting transgenes to the endogenous Rosa26 promoter results in moderate ubiquitous expression and is not suitable for high expression levels. Therefore, we now generated a modified Rosa26 (modRosa26) locus that combines efficient targeted transgenesis using recombinase-mediated cassette exchange (RMCE) by Flipase (Flp-RMCE) or Cre recombinase (Cre-RMCE) with transgene expression from exogenous promoters. We silenced the endogenous Rosa26 promoter and characterized several ubiquitous (pCAG, EF1α and CMV) and tissue-specific (VeCad, αSMA) promoters in the modRosa26 locus in vivo. We demonstrate that the ubiquitous pCAG promoter in the modRosa26 locus now offers high transgene expression. While tissue-specific promoters were all active in their cognate tissues they additionally led to rare ectopic expression. To achieve high expression levels in a tissue-specific manner, we therefore combined Flp-RMCE for rapid ES cell targeting, the pCAG promoter for high transgene levels and Cre/LoxP conditional transgene activation using well-characterized Cre lines. Using this approach we generated a Cre/LoxP-inducible reporter mouse line with high EGFP expression levels that enables cell tracing in live cells. A second reporter line expressing luciferase permits efficient monitoring of Cre activity in live animals. Thus, targeting the modRosa26 locus by RMCE minimizes the effort required to target ES cells and generates a tool for the use exogenous promoters in combination with single-copy transgenes for predictable expression in mice

The proton-activated receptor GPR4 contributes to central respiratory chemosensitivity

The ability to sense CO2/H+ is critical for normal breathing. A specific group of neurons within the medullary retrotrapezoid nucleus (RTN), identified by Phox2b-expression, are intrinsically sensitive to CO2/H+ and are preeminent central respiratory chemosensory neurons1,2. These excitatory neurons stimulate ventilation to rapidly regulate CO2 excretion and acid-base balance3 and they are selectively ablated in a mouse genetic model of human congenital central hypoventilation syndrome (CCHS) that recapitulates the blunted CO2-induced breathing and increased central apneas of CCHS4-6. However, the molecular mechanisms that mediate pH sensing in these neurons remain incompletely understood. Here, we demonstrate that GPR4, a proton-activated G protein-coupled receptor, represents a molecular substrate for CO2/H+-dependent regulation of RTN neuronal excitability and central respiratory chemosensitivity. Ventilatory stimulation by raised CO2, but not lowered O2, was strongly reduced in GPR4 knockout mice, which were also more prone to apneic events during quiet hyperoxic breathing. Mice lacking GPR4 showed a striking reduction in CO2 activation of RTN neurons in vivo, as determined by cFos expression. Likewise, RTN neuronal excitability and pH sensitivity in vitro were reduced by GPR4 deletion or receptor blockade, and modulated by intracellular application of GTP analogs. Finally, re-expression of GPR4 into RTN neurons of GPR4-/- mice rescued CO2-stimulated cFos expression and ventilation, and returned apneic frequencies to wild type levels. Taken together, this work identifies a new role for GPR4, defines a novel molecular component of central respiratory chemosensitivity, and suggests new therapeutic options to regulate breathing

The G protein-coupled pH-sensing receptor OGR1 is a regulator of intestinal inflammation

Background: A novel family of proton sensing G-protein coupled receptors (GPCRs), including ovarian cancer G-protein coupled receptor 1 (OGR1, GPR68), GPR4, and T-cell death associated gene 8 (TDAG8, GPR65), was previously identified to play an important role in physiological pH homeostasis. Luminal and tissue pH values in patients with active inflammatory bowel disease (IBD) are known to be significantly lower than values in the normal subject. We investigated whether OGR1 may play a role for the pathophysiology of IBD. Methods: OGR1 expression in colonic tissues was investigated in controls and IBD patients. Expression of OGR1 upon cell activation was studied in the Mono Mac 6 (MM6) cell line and primary human and murine monocytes by real-time PCR. Ogr1 knockout mice were crossbred with Il-10 deficient mice, compared to Il-10 -/- from the same litters and studied over 200 days. Microarray profiling (Metacore GeneGo and ranked fold change analysis) was performed using Ogr1-/- and Ogr1+/+ (WT) residential peritoneal macrophages. Results: IBD patients expressed higher levels of OGR1 in the mucosa as compared to controls. Treatment of MM6 cells with TNF, but not other cytokines (IFN-γ, IL-1β, IL-6, TGF-β) tested, led to significant up-regulation of OGR1 mRNA expression. Induction of OGR1 expression by TNF was dose-dependent. Macrophage differentiation of MM6 cells with PMA also led to a significant increase in OGR1 expression. Dose-dependence of TNF and PMA mediated induction of OGR1 expression was confirmed in primary human monocytes. TNF-mediated induction of OGR1 mRNA expression was reversed by simultaneous treatment of cells with the NF-κB inhibitors MG132, AICAR, BAY-11-7082, CAY10512, and SC-514. Kaplan-Meier survival analysis showed a significantly delayed onset and progression of rectal prolapse in female Ogr1-/- // Il-10-/- mice (p=0.005). Female Ogr1 -/- // Il-10 -/- mice had significantly less rectal prolapses (21%, n = 19 versus 75%, n = 8; p = 0.025; Odd ratio = 0.089). Up-regulation of gene expression, mediated by OGR1, in response to extracellular acidification in mouse macrophages was enriched for inflammation and immune response, actin cytoskeleton, and cell adhesion gene pathways. Conclusion: OGR1 expression is induced in cells of human macrophage lineage and primary human monocytes by TNF and PMA. NF-κB inhibition reverses the induction of OGR1 mRNA expression by TNF. OGR1 deficiency protects from spontaneous inflammation in the Il-10 KO model. Our data indicate an important pathophysiological role for the pH sensing G-protein coupled receptor OGR1 during the pathogenesis of mucosal inflammation

Effects of the fibroblast activation protein inhibitor, PT100, in a murine model of pulmonary fibrosis

Bleomycin (BLM) induced lung injury is detectable in C57BL/6 mice using magnetic resonance imaging (MRI). We investigated the effects of the fibroblast activation protein (FAP) inhibitor, PT100, in this model. BLM (0.5 mg/kg/day) was administered on days −7, −6, −5, −2, −1, 0 in the nostrils of male mice. PT100 (40 µg/mouse) or vehicle (0.9%NaCl) was dosed per os twice daily from day 1–14. MRI was performed before BLM and at days 0, 7 and 14. After the last MRI acquisition, animals were euthanised and the lungs harvested for histological and quantitative real-time polymerase chain reaction (qRT-PCR) analyses. As evidenced longitudinally by MRI, the BLM-elicited lesions in the lungs of vehicle-treated mice progressed over time. In contrast, responses elicited by BLM did not progress in animals receiving PT100. Histology demonstrated significant less fibrosis in PT100- than in vehicle-treated, BLM-challenged mice. Significant correlation (R=0.91, P<0.001, N=24) was found between the volumes of BLM-induced lesions detected in vivo by MRI and the collagen content determined histologically (picrosirius staining). FAP was overexpressed in the lungs of BLM-challenged mice. Upon PT100 treatment, FAP expression was reduced. Significant differences in the MMP-12, MIP-1α, and MCP-3 mRNA expression levels in the lungs of PT100- compared to vehicle-treated mice were also revealed by qRT-PCR. The IBA-1 level determined histologically was higher in the lungs of PT100- compared to vehicle-treated mice. Taken together, these observations suggest that treatment with PT100 in this murine model of pulmonary fibrosis had an anti-fibro-proliferative effect and increased macrophage activation

Neuropeptide S receptor deficiency modulates spontaneous locomotor activity and the acoustic startle response

The present study investigated the phenotype of heterozygous and homozygous neuropeptide S receptor (Npsr) deficient C57BL/6 mice in NPS- and cocaine induced hyperactivity, spontaneous and reactive locomotor activity, elevated plus maze, conditioned fear, and prepulse inhibition of the acoustic startle response. In Npsr-deficient mice, a strong reduction of spontaneous locomotor activity and of the startle magnitude was observed; heterozygous mice had an intermediate phenotype. In the other experiments, Npsr deficiency leads to no or only a very modest phenotype. These results support an important role of neuropeptide S in regulating locomotor activity

The Proton-Activated Receptor GPR4 Modulates Glucose Homeostasis by Increasing Insulin Sensitivity

Background: The proton-activated G protein-coupled receptor GPR4 is expressed in many tissues including white adipose tissue. GPR4 is activated by extracellular protons in the physiological pH range (i.e. pH 7.7 - 6.8) and is coupled to the production of cAMP. Methods: We examined mice lacking GPR4 and examined glucose tolerance and insulin sensitivity in young and aged mice as well as in mice fed with a high fat diet. Expression profiles of pro- and anti-inflammatory cytokines in white adipose tissue, liver and skeletal muscle was assessed. Results: Here we show that mice lacking GPR4 have an improved intraperitoneal glucose tolerance test and increased insulin sensitivity. Insulin levels were comparable but leptin levels were increased in GPR4 KO mice. Gpr4-/- showed altered expression of PPARα, IL-6, IL-10, TNFα, and TGF-1β in skeletal muscle, white adipose tissue, and liver. High fat diet abolished the differences in glucose tolerance and insulin sensitivity between Gpr4+/+ and Gpr4-/- mice. In contrast, in aged mice (12 months old), the positive effect of GPR4 deficiency on glucose tolerance and insulin sensitivity was maintained. Liver and adipose tissue showed no major differences in the mRNA expression of pro- and anti-inflammatory factors between aged mice of both genotypes. Conclusion: Thus, GPR4 deficiency improves glucose tolerance and insulin sensitivity. The effect may involve an altered balance between pro- and anti-inflammatory factors in insulin target tissues

Administration of bleomycin via the oropharyngeal aspiration route leads to sustained lung fibrosis in mice and rats as quantified by UTE-MRI and histology.

Pulmonary fibrosis can be experimentally induced in small rodents by bleomycin. The antibiotic is usually administered via the intratracheal or intranasal routes. In the present study, we investigated the oropharyngeal aspiration of bleomycin as an alternative route for the induction of lung fibrosis in rats and mice. The development of lung injury was followed in vivo by ultrashort echo time magnetic resonance imaging (UTE-MRI) and by post-mortem analyses (histology of collagen, hydroxyproline determination, and qRT-PCR). In C57BL/6 mice, oropharyngeal aspiration of bleomycin led to more prominent lung fibrosis as compared to intranasal administration. Consequently, the oropharyngeal aspiration route allowed a dose reduction of bleomycin and, therewith, a model refinement. Moreover, the distribution of collagen after oropharyngeal aspiration of bleomycin was more homogenous than after intranasal administration: for the oropharyngeal aspiration route, fibrotic areas appeared all over the lung lobes, while for the intranasal route fibrotic lesions appeared mainly around the largest superior airways. Thus, oropharyngeal aspiration of bleomycin induced morphological changes that were more comparable to the human disease than the intranasal administration route did. Oropharyngeal aspiration of bleomycin led to a homogeneous fibrotic injury also in rat lungs. The present data suggest oropharyngeal aspiration of bleomycin as a less invasive means to induce homogeneous and sustained fibrosis in the lungs of mice and rats

Correlations between MRI and all other readouts on day 23 analyzed in the present study for detection of bleomycin-induced lung fibrosis in BALB/c mice (n = 32; except for Picrosirius n = 16): total MRI signal volume, picrosirius staining, hydroxyproline, and gene expression of Col1α1, F4/80 and MMP12.

<p>Saline-treated BALB/c animals (n = 8, n = 4 for picrosirius) were used as controls.</p