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BACKGROUND: Prenatal glucocorticoid treatment decreases alveolar tissue volumes and facilitates fetal lung maturation, however the mechanisms responsible are largely unknown. This study examines whether changes in versican levels or sulphation patterns of chondroitin sulphate (CS) side chains, are associated with glucocorticoid-induced reductions in peri-alveolar tissue volumes. METHODS: Lung tissue was collected from 1) fetal sheep at 131 ± 0.1 days gestational age (GA) infused with cortisol (122-131d GA) to prematurely induce a pre-parturient-like rise in circulating cortisol, 2) fetal sheep at 143d GA bilaterally adrenalectomised (ADX) at 112d GA to remove endogenous cortisol and 3) fetal sheep at 124d GA in which bolus doses (2 × 11.4 mg) of betamethasone were administered to the pregnant ewe. The level and distribution of versican and CS glycosaminoglycans (GAG) were determined using immunohistochemistry (IHC). Fluorophore assisted carbohydrate electrophoresis (FACE) was used to determine changes in CS sulphation patterns. RESULTS: Cortisol infusion significantly decreased chondrotin-6-sulphate levels (C-6-S) to 16.4 ± 0.7 AU, compared with saline-infused fetuses (18.9 ± 0.7 AU: p = 0.04) but did not significantly alter the level of versican or chondroitin-4-sulphate (C-4-S). ADX significantly increased the level of C-4-S (28.2 ± 2.2 AU), compared with sham-operated fetuses (17.8 ± 2.0 AU; p = 0.006) without altering versican or C-6-S levels. Betamethasone significantly decreased versican, C-4-S and C-6-S in the fetal sheep lung (19.2 ± 0.9 AU, 24.9 ± 1.4 AU and 23.2 ± 1.0 AU, respectively), compared with saline-exposed fetuses (24.3 ± 0.4 AU, p = 0.0004; 33.3±0.6 AU, p = 0.0003; 29.8±1.3 AU, 0.03, respectively). CONCLUSIONS: These results indicate that glucocorticoids alter versican levels and CS side chain microstructure in alveolar lung tissue. Betamethasone appears to have a greater impact on versican and CS side chains than cortisol.

© 2017 The Author(s). Cigarette smoking is undoubtedly a risk factor for lung cancer. Moreover, smokers with genetic mutations on chromosome 3p21.3, a region frequently deleted in cancer and notably in lung cancer, have a dramatically higher risk of aggressive lung cancer. The RNA binding motif 5 (RBM5) is one of the component genes in the 3p21.3 tumour suppressor region. Studies using human cancer specimens and cell lines suggest a role for RBM5 as a tumour suppressor. Here we demonstrate, for the first time, an in vivo role for RBM5 as a tumour suppressor in the mouse lung. We generated Rbm5 loss-of-function mice and exposed them to a tobacco carcinogen NNK. Upon exposure to NNK, Rbm5 loss-of-function mice developed lung cancer at similar rates to wild type mice. As tumourigenesis progressed, however, reduced Rbm5 expression lead to significantly more aggressive lung cancer i.e. increased adenocarcinoma nodule numbers and tumour size. Our data provide in vivo evidence that reduced RBM5 function, as occurs in a large number of patients, coupled with exposure to tobacco carcinogens is a risk factor for an aggressive lung cancer phenotype. These data suggest that RBM5 loss-of-function likely underpins at least part of the pro-tumourigenic consequences of 3p21.3 deletion in humans.

Treatment of preterm human infants with high oxygen can result in disrupted lung alveolar and vascular development. Local or systemic administration of endothelial progenitor cells (EPCs) is reported to remedy such disruption in animal models. In this study, the effects of both fresh (enriched for KDR) and cultured bone marrow (BM)-derived cell populations with EPC characteristics were examined following hyperoxia in neonatal mouse lungs. Intraperitoneal injection of fresh EPCs into five-day-old mice treated with 90% oxygen resulted in full recovery of hyperoxia-induced alveolar disruption by 56 days of age. Partial recovery in septal number following hyperoxia was observed following injection of short-term cultured EPCs, yet aberrant tissue growths appeared following injection of long-term cultured cells. Fresh and long-term cultured cells had no impact on blood vessel development. Short-term cultured cells increased blood vessel number in normoxic and hyperoxic mice by 28 days but had no impact on day 56. Injection of fresh EPCs into normoxic mice significantly reduced alveolarization compared with phosphate buffered saline-injected normoxic controls. These results indicate that fresh BM EPCs have a higher and safer corrective profile in a hyperoxia-induced lung injury model compared with cultured BM EPCs but may be detrimental to the normoxic lung. The appearance of aberrant tissue growths and other side effects following injection of cultured EPCs warrants further investigation. Stem Cells Translational Medicine 2017;6:2094-2105.

During mammalian lung development, the morphological transition from respiratory tree branching morphogenesis to a predominantly saccular architecture, capable of air-breathing at birth, is dependent on physical forces as well as molecular signaling by a range of transcription factors including the cAMP response element binding protein 1 (Creb1). Creb1(-/-) mutant mice exhibit complete neonatal lethality consistent with a lack of lung maturation beyond the branching phase. To further define its role in the developing mouse lung, we deleted Creb1 separately in the respiratory epithelium and mesenchyme. Surprisingly, we found no evidence of a morphological lung defect nor compromised neonatal survival in either conditional Creb1 mutant. Interestingly however, loss of mesenchymal Creb1 on a genetic background lacking the related Crem protein showed normal lung development but poor neonatal survival. To investigate the underlying requirement for Creb1 for normal lung development, Creb1(-/-) mice were re-examined for defects in both respiratory muscles and glucocorticoid hormone signaling, which are also required for late stage lung maturation. However, these systems appeared normal in Creb1(-/-) mice. Together our results suggest that the requirement of Creb1 for normal mammalian lung morphogenesis is not dependent upon its expression in lung epithelium or mesenchyme, nor its role in musculoskeletal development.

BACKGROUND: Mice harbouring gene mutations that cause phenotypic abnormalities during organogenesis are invaluable tools for linking gene function to normal development and human disorders. To generate mouse models harbouring novel alleles that are involved in organogenesis we conducted a phenotype-driven, genome-wide mutagenesis screen in mice using the mutagen N-ethyl-N-nitrosourea (ENU). METHODOLOGY/PRINCIPAL FINDINGS: ENU was injected into male C57BL/6 mice and the mutations transmitted through the germ-line. ENU-induced mutations were bred to homozygosity and G3 embryos screened at embryonic day (E) 13.5 and E18.5 for abnormalities in limb and craniofacial structures, skin, blood, vasculature, lungs, gut, kidneys, ureters and gonads. From 52 pedigrees screened 15 were detected with anomalies in one or more of the structures/organs screened. Using single nucleotide polymorphism (SNP)-based linkage analysis in conjunction with candidate gene or next-generation sequencing (NGS) we identified novel recessive alleles for Fras1, Ift140 and Lig1. CONCLUSIONS/SIGNIFICANCE: In this study we have generated mouse models in which the anomalies closely mimic those seen in human disorders. The association between novel mutant alleles and phenotypes will lead to a better understanding of gene function in normal development and establish how their dysfunction causes human anomalies and disease.

The cAMP response element binding protein 1 (Creb1) transcription factor regulates cellular gene expression in response to elevated levels of intracellular cAMP. Creb1(-/-) fetal mice are phenotypically smaller than wildtype littermates, predominantly die in utero and do not survive after birth due to respiratory failure. We have further investigated the respiratory defect of Creb1(-/-) fetal mice during development. Lungs of Creb1(-/-) fetal mice were pale in colour and smaller than wildtype controls in proportion to their reduced body size. Creb1(-/-) lungs also did not mature morphologically beyond E16.5 with little or no expansion of airway luminal spaces, a phenotype also observed with the Creb1(-/-) lung on a Crem(-/-) genetic background. Creb1 was highly expressed throughout the lung at all stages examined, however activation of Creb1 was detected primarily in distal lung epithelium. Cell differentiation of E17.5 Creb1(-/-) lung distal epithelium was analysed by electron microscopy and showed markedly reduced numbers of type-I and type-II alveolar epithelial cells. Furthermore, immunomarkers for specific lineages of proximal epithelium including ciliated, non-ciliated (Clara), and neuroendocrine cells showed delayed onset of expression in the Creb1(-/-) lung. Finally, gene expression analyses of the E17.5 Creb1(-/-) lung using whole genome microarray and qPCR collectively identified respiratory marker gene profiles and provide potential novel Creb1-regulated genes. Together, these results demonstrate a crucial role for Creb1 activity for the development and differentiation of the conducting and distal lung epithelium.

An age-dependent association between variation at the FTO locus and BMI in children has been suggested. We meta-analyzed associations between the FTO locus (rs9939609) and BMI in samples, aged from early infancy to 13 years, from 8 cohorts of European ancestry. We found a positive association between additional minor (A) alleles and BMI from 5.5 years onwards, but an inverse association below age 2.5 years. Modelling median BMI curves for each genotype using the LMS method, we found that carriers of minor alleles showed lower BMI in infancy, earlier adiposity rebound (AR), and higher BMI later in childhood. Differences by allele were consistent with two independent processes: earlier AR equivalent to accelerating developmental age by 2.37% (95% CI 1.87, 2.87, p = 10(-20)) per A allele and a positive age by genotype interaction such that BMI increased faster with age (p = 10 223). We also fitted a linear mixed effects model to relate genotype to the BMI curve inflection points adiposity peak (AP) in infancy and AR. Carriage of two minor alleles at rs9939609 was associated with lower BMI at AP (-0.40% (95% CI: -0.74, -0.06), p = 0.02), higher BMI at AR (0.93% (95% CI: 0.22, 1.64), p = 0.01), and earlier AR (-4.72% (-5.81, -3.63), p = 10 217), supporting cross-sectional results. Overall, we confirm the expected association between variation at rs9939609 and BMI in childhood, but only after an inverse association between the same variant and BMI in infancy. Patterns are consistent with a shift on the developmental scale, which is reflected in association with the timing of AR rather than just a global increase in BMI. Results provide important information about longitudinal gene effects and about the role of FTO in adiposity. The associated shifts in developmental timing have clinical importance with respect to known relationships between AR and both later-life BMI and metabolic disease risk.

Zn²(+)-finger proteins comprise one of the largest protein superfamilies with diverse biological functions. The ATM substrate Chk2-interacting Zn²(+)-finger protein (ASCIZ; also known as ATMIN and ZNF822) was originally linked to functions in the DNA base damage response and has also been proposed to be an essential cofactor of the ATM kinase. Here we show that absence of ASCIZ leads to p53-independent late-embryonic lethality in mice. Asciz-deficient primary fibroblasts exhibit increased sensitivity to DNA base damaging agents MMS and H2O2, but Asciz deletion knock-down does not affect ATM levels and activation in mouse, chicken, or human cells. Unexpectedly, Asciz-deficient embryos also exhibit severe respiratory tract defects with complete pulmonary agenesis and severe tracheal atresia. Nkx2.1-expressing respiratory precursors are still specified in the absence of ASCIZ, but fail to segregate properly within the ventral foregut, and as a consequence lung buds never form and separation of the trachea from the oesophagus stalls early. Comparison of phenotypes suggests that ASCIZ functions between Wnt2-2b/ß-catenin and FGF10/FGF-receptor 2b signaling pathways in the mesodermal/endodermal crosstalk regulating early respiratory development. We also find that ASCIZ can activate expression of reporter genes via its SQ/TQ-cluster domain in vitro, suggesting that it may exert its developmental functions as a transcription factor. Altogether, the data indicate that, in addition to its role in the DNA base damage response, ASCIZ has separate developmental functions as an essential regulator of respiratory organogenesis.

BACKGROUND: Bronchopulmonary dysplasia (BPD) is closely associated with ventilator-induced lung injury (VILI) in very preterm infants. The greatest risk of VILI may be in the immediate period after birth, when the lungs are surfactant deficient, still partially filled with liquid and not uniformly aerated. However, there have been very few studies that have examined this immediate post-birth period and identified the initial injury-related pathways that are activated. We aimed to determine if the early response genes; connective tissue growth factor (CTGF), cysteine rich-61 (CYR61) and early growth response 1 (EGR1), were rapidly induced by VILI in preterm lambs and whether ventilation with different tidal volumes caused different inflammatory cytokine and early response gene expression. METHODS: To identify early markers of VILI, preterm lambs (132 d gestational age; GA, term approximately 147 d) were resuscitated with an injurious ventilation strategy (V(T) 20 mL/kg for 15 min) then gently ventilated (5 mL/kg) for 15, 30, 60 or 120 min (n = 4 in each). To determine if early response genes and inflammatory cytokines were differentially regulated by different ventilation strategies, separate groups of preterm lambs (125 d GA; n = 5 in each) were ventilated from birth with a V(T) of 5 (VG5) or 10 mL/kg (VG10) for 135 minutes. Lung gene expression levels were compared to levels prior to ventilation in age-matched control fetuses. RESULTS: CTGF, CYR61 and EGR1 lung mRNA levels were increased approximately 25, 50 and 120-fold respectively (p < 0.05), within 30 minutes of injurious ventilation. VG5 and VG10 caused significant increases in CTGF, CYR61, EGR1, IL1- , IL-6 and IL-8 mRNA levels compared to control levels. CTGF, CYR61, IL-6 and IL-8 expression levels were higher in VG10 than VG5 lambs; although only the IL-6 and CYR61 mRNA levels reached significance. CONCLUSION: CTGF, CYR61 and EGR1 may be novel early markers of lung injury and mechanical ventilation from bir

T lymphocytes (pivotal in many inflammatory pathologies) are targets for glucocorticoid hormone (GC). How TCR-mediated activation and GC signaling via glucocorticoid receptor (GR) impact on T-cell fates is not fully defined. We delineated here the expression of a recently identified glucocorticoid-induced TNF receptor (GITR) induced by GC and by TCR-mediated T-cell activation in GC receptor (GR)-deficient mice (GR-/-). We also compared the action of GC on GITR+ and GITR- T cells by monitoring apoptosis, proliferation and cytokine production stimulated by anti-CD3 antibody. By using GR-/- mice, we observed that the development of GITR+ T cells (both in thymus and periphery) is not dependent upon GR signaling. This contradicts the implication of GITR's name reflecting GC induction. TCR-mediated T-cell activation induced GITR expression in both GR+/+ and GR-/- cells. Somewhat unexpectedly, there was very modest GITR upregulation on GR+/+ T cells by a range of GC doses (10(-8) to 10(-6) M). Constitutive expression of GITR by a subset of CD4+ cells did not significantly render them resistant to GC-induced cell death. However, TCR-induced GITR upregulation on GR+/+ T cells was correlated with resistance to GC-mediated apoptosis suggesting that GITR, in conjunction with other (as yet unidentified) TCR-induced factors, protects T cells from apoptosis. Thus, even though GC is a potent inducer of apoptosis of T cells, activated T cells are resistant to GC-mediated killing. Meanwhile, although GC suppressed anti-CD3-induced cytokine production, cell proliferation was unaffected by GC in GR+/+ mice. GR deficiency has no effect on anti-CD3-induced cytokine production and proliferation. Our findings also have implications for GC treatment in that it would be more difficult to abrogate an ongoing T-cell mediated inflammatory response than to prevent its induction.

Peripheral activation can cause bystander thymocyte death by eliciting a "cytokine storm." This event complicates in vivo studies using exogenous ligand-induced models of negative selection. A stable transgenic model that selectively eliminates peripheral CD4 cells has allowed us to analyze negative selection as direct cognate events in two T cell receptor transgenic mice, OT-II and DO11. Whereas cognate peptide induced a massive deletion in double-positive (DP) cells in mice with peripheral CD4 cells, this DP deletion was modest in mice lacking peripheral CD4 cells. Using BrdUrd and annexin V staining, we found that negative selection primarily occurs in a cohort of DP cells and the absence of single-positive (SP) cells is largely caused by reduction in the cohort of DP precursors. Moreover, the fates of DP cells and SP cells after antigen exposure were vastly different. Whereas SP cells up-regulated uniformly their CD69 and CD44 levels, increased their cell size, and survived after antigen exposure, DP cells had less CD69 and CD44 up-regulation, no size change, and promptly died. Thus, negative selection represents an "abortive" activation different from activation-induced cell death of mature T cells.

Ca(2+) signaling plays a central role in cardiac contractility and adaptation to increased hemodynamic demand. We have generated mice with a targeted deletion of the S100A1 gene coding for the major cardiac isoform of the large multigenic S100 family of EF hand Ca(2+)-binding proteins. S100A1(-/-) mice have normal cardiac function under baseline conditions but have significantly reduced contraction rate and relaxation rate responses to beta-adrenergic stimulation that are associated with a reduced Ca(2+) sensitivity. In S100A1(-/-) mice, basal left-ventricular contractility deteriorated following 3-week pressure overload by thoracic aorta constriction despite a normal adaptive hypertrophy. Surprisingly, heterozygotes also had an impaired response to acute beta-adrenergic stimulation but maintained normal contractility in response to chronic pressure overload that coincided with S100A1 upregulation to wild-type levels. In contrast to other genetic models with impaired cardiac contractility, loss of S100A1 did not lead to cardiac hypertrophy or dilation in aged mice. The data demonstrate that high S100A1 protein levels are essential for the cardiac reserve and adaptation to acute and chronic hemodynamic stress in vivo.

The involvement of glucocorticoid receptor (GR) signaling in T cell development is highly controversial, with several studies for and against. We have previously demonstrated that GR(-/-) mice, which usually die at birth because of impaired lung development, exhibit normal T cell development, at least in embryonic mice and in fetal thymus organ cultures. To directly investigate the role of GR signaling in adult T cell development, we analyzed the few GR(-/-) mice that occasionally survive birth, and irradiated mice reconstituted with GR(-/-) fetal liver precursors. All thymic and peripheral T cells, as well as other leukocyte lineages, developed and were maintained at normal levels. Anti-CD3-induced cell death of thymocytes in vitro, T cell repertoire heterogeneity and T cell proliferation in response to anti-CD3 stimulation were normal in the absence of GR signaling. Finally, we show that metyrapone, an inhibitor of glucocorticoid synthesis (commonly used to demonstrate a role for glucocorticoids in T cell development), impaired thymocyte development regardless of GR genotype indicating that this reagent inhibits thymocyte development in a glucocorticoid-independent fashion. These data demonstrate that GR signaling is not required for either normal T cell development or peripheral maintenance in embryonic or adult mice.