Claude Jourdan Le Saux

The LOXL2 Gene Encodes a New Lysyl Oxidase-like Protein and Is Expressed at High Levels in Reproductive Tissues

We have reported in this paper the complete cDNA sequence, gene structure, and tissue-specific expression of LOXL2, a new amine oxidase and a member of an emerging family of human lysyl oxidases. The predicted amino acid sequence, from several overlapping cDNA clones isolated from placenta and spleen cDNA libraries, shared extensive sequence homology with the conserved copper-binding and catalytic domains of both lysyl oxidase (LOX) and the lysyl oxidase-like (LOXL) protein. These conserved domains are encoded by five consecutive exons within the LOX,LOXL, and LOXL2 genes that also maintained exon-intron structure conservation. In contrast, six exons encoding the amino-terminal domains diverged both in sequence and structure. Exon 1 of the LOXL2 gene does not encode a signal sequence that is present in LOX and LOXL, suggesting a different processing and intracellular localization for this new protein. Expression of the LOXL2 gene was detected in almost all tissues with the highest steady state mRNA levels in the reproductive tissues, placenta, uterus and prostate. In situ hybridization identified placental syncytial and cytotrophoblasts responsible for the synthesis of LOXL2mRNA and demonstrated a spatial and temporal expression pattern unique to the LOXL2 gene.

Cellular senescence increases expression of bacterial ligands in the lungs and is positively correlated with increased susceptibility to pneumococcal pneumonia

Cellular senescence is an age‐associated phenomenon that promotes tumor invasiveness owing to the secretion of proinflammatory cytokines, proteases, and growth factors. Herein we demonstrate that cellular senescence also potentially increases susceptibility to bacterial pneumonia caused by Streptococcus pneumoniae (the pneumococcus), the leading cause of infectious death in the elderly. Aged mice had increased lung inflammation as determined by cytokine analysis and histopathology of lung sections. Immunoblotting for p16, pRb, and mH2A showed that elderly humans and aged mice had increased levels of these senescence markers in their lungs vs. young controls. Keratin 10 (K10), laminin receptor (LR), and platelet‐activating factor receptor (PAFr), host proteins known to be co‐opted for bacterial adhesion, were also increased. Aged mice were found to be highly susceptible to pneumococcal challenge in a PsrP, the pneumococcal adhesin that binds K10, dependent manner. In vitro senescent A549 lung epithelial cells had elevated K10 and LR protein levels and were up to 5‐fold more permissive for bacterial adhesion. Additionally, exposure of normal cells to conditioned media from senescent cells doubled PAFr levels and pneumococcal adherence. Genotoxic stress induced by bleomycin and oxidative stress enhanced susceptibility of young mice to pneumonia and was positively correlated with enhanced p16, inflammation, and LR levels. These findings suggest that cellular senescence facilitates bacterial adhesion to cells in the lungs and provides an additional molecular mechanism for the increased incidence of community‐acquired pneumonia in the elderly. This study is the first to suggest a second negative consequence for the senescence‐associated secretory phenotype.

A role for dietary selenium and selenoproteins in allergic airway inflammation.

Asthma is driven by allergic airway inflammation and involves increased levels of oxidative stress. This has led to speculation that antioxidants like selenium (Se) may play important roles in preventing or treating asthma. We fed diets containing low (0.08 parts per million), medium (0.25 parts per million), or high (2.7 parts per million) Se to female C57BL/6 mice and used an established OVA challenge protocol to determine the relationship between Se intake and the development of allergic airway inflammation. Results demonstrated that mice fed medium levels of Se had robust responses to OVA challenge in the lung as measured by lung cytokine levels, airway cellular infiltrate, eosinophilia, serum anti-OVA IgE, airway hyperreactivity, goblet cell hyperplasia, and phosphorylated STAT-6 levels in the lung. In contrast, responses to OVA challenge were less robust in mice fed low or high levels of Se. In particular, mice fed low Se chow showed significantly lower responses compared with mice fed medium Se chow for nearly all readouts. We also found that within the medium Se group the expression of lung glutathione peroxidase-1 and liver selenoprotein P were increased in OVA-challenged mice compared with PBS controls. These data suggest that Se intake and allergic airway inflammation are not related in a simple dose-response manner, which may explain the inconsistent results obtained from previous descriptive studies in humans. Also, our results suggest that certain selenoproteins may be induced in response to Ag challenges within the lung.

Down-regulation of Caveolin-1, an Inhibitor of Transforming Growth Factor-β Signaling, in Acute Allergen-induced Airway Remodeling

Asthma can progress to subepithelial airway fibrosis, mediated in large part by transforming growth factor-β (TGF-β). The scaffolding protein caveolin-1 (cav1) can inhibit the activity of TGF-β, perhaps by forming membrane invaginations that enfold TGF-β receptors. The study goals were 1) to evaluate how allergen challenge affects lung expression of cav1 and the density of caveolae in vivo 2) to determine whether reduced cav1 expression is mediated by interleukin (IL)-4 and 3) to measure the effects of decreased expression of cav1 on TGF-β signaling. C57BL/6J, IL-4-deficient mice, and cav1-deficient mice, sensitized by intraperitoneal injections of phosphate-buffered saline or ovalbumin (OVA) at days 0 and 12, received intranasal phosphate-buffered saline or OVA challenges at days 24, 26, and 28. Additionally, another group of C57BL/6J mice received IL-4 by intratracheal instillation for 7 days. We confirmed that the OVA-allergen challenge increased eosinophilia and T-helper type 2-related cytokine levels (IL-4, IL-5, and IL-13) in bronchoalveolar lavage. Allergen challenge reduced lung cav1 mRNA abundance by 40%, cav1 protein by 30%, and the number of lung fibroblast caveolae by 50%. Administration of IL-4 in vivo also substantially decreased cav1 expression. In contrast, the allergen challenge did not decrease cav1 expression in IL-4-deficient mice. The reduced expression of cav1 was associated with activation of TGF-β signaling that was further enhanced in OVA-sensitized and challenged cav1-deficient mice. This study demonstrates a previously unknown modulation of TGF-β signaling by IL-4, via cav1, suggesting novel therapeutic targets for controlling the effects of TGF-β and thereby ameliorating pathological airway remodeling.

Caveolin-1 Deficiency Protects from Pulmonary Fibrosis by Modulating Epithelial Cell Senescence in Mice

Idiopathic pulmonary fibrosis is associated with a decreased expression of caveolin-1 (cav-1), yet its role remains unclear. To investigate the role of cav-1, we induced pulmonary fibrosis in wild-type (WT) and cav-1-deficient (cav-1(-/-)) mice using intratracheal instillation of bleomycin. Contrary to expectations, significantly less collagen deposition was measured in tissue from cav-1(-/-) mice than in their WT counterparts, consistent with reduced mRNA expression of procollagen1a2 and procollagen3a1. Moreover, cav-1(-/-) mice demonstrated 77% less α-smooth muscle actin staining, suggesting reduced mesenchymal cell activation. Levels of pulmonary injury, assessed by tenascin-C mRNA expression and CD44v10 detection, were significantly increased at Day 21 after injury in WT mice, an effect significantly attenuated in cav-1(-/-) mice. The apparent protective effect against bleomycin-induced fibrosis in cav-1(-/-) mice was attributed to reduce cellular senescence and apoptosis in cav-1(-/-) epithelial cells during the early phase of lung injury. Reduced matrix metalloproteinase (MMP)-2 and MMP-9 expressions indicated a low profile of senescence-associated secretory phenotype (SASP) in the bleomycin-injured cav-1(-/-) mice. However, IL-6 and macrophage inflammatory protein 2 were increased in WT and cav-1(-/-) mice after bleomycin challenge, suggesting that bleomycin-induced inflammatory response substantiated the SASP pool. Thus, loss of cav-1 attenuates early injury response to bleomycin by limiting stress-induced cellular senescence/apoptosis in epithelial cells. In contrast, decreased cav-1 expression promotes fibroblast activation and collagen deposition, effects that may be relevant in later stages of reparative response. Hence, therapeutic strategies to modulate the expression of cav-1 should take into account cell-specific effects in the regenerative responses of the lung epithelium to injury.

Caveolin-1 modulates TGF-β1 signaling in cardiac remodeling

The cardiac response to myocardial injury includes fibrotic and hypertrophic processes and a key mediator in this response is transforming growth factor-β1 (TGF-β1). Caveolin-1 (cav1), the main structural protein of caveolae, is an inhibitor of the TGF-β1 signaling pathway. To examine the role of cav1 in cardiac repair, cav1 deficient (Cav1(-/-)) and wild type (WT) mice were subjected to cryoinjury of the left ventricle (LV). At baseline the two groups exhibited no inflammation, similar collagen content, and similar cardiac function. After injury, Cav1(-/-) animals displayed enhanced TGF-β1 signaling, as reflected by a 3-fold increase in the activation of the Smad2-dependent pathway and more widespread collagen deposition in the heart. Qualitative and quantitative analyses indicated that collagen deposition peaked in the WT LV 14days after injury, accompanied by increased mRNA abundance for procol1a2 (2-fold) and procol3a1 (3-fold). Collagen deposition was further enhanced in Cav1(-/-) mice, which was accompanied by reduced expression of matrix metalloproteinases MMP-8 (3-fold) and -13 mRNA (2-fold). The levels of expression of inflammatory markers of acute phase were similar between the strains However, macrophage clearance in the damaged region was delayed in Cav1(-/-) mice. We observed a 4-fold decrease in collagen deposition in Cav1(-/-) mice injected with a cav1 scaffolding domain peptide (CSD) and a 2-fold decrease in WT mice treated with the CSD. We conclude that cav1 has a direct role in reducing TGF-β1 signaling and as such might be an appropriate target for therapies to influence cardiac remodeling.

A Novel Telomerase Activator Suppresses Lung Damage in a Murine Model of Idiopathic Pulmonary Fibrosis

The emergence of diseases associated with telomere dysfunction, including AIDS, aplastic anemia and pulmonary fibrosis, has bolstered interest in telomerase activators. We report identification of a new small molecule activator, GRN510, with activity ex vivo and in vivo. Using a novel mouse model, we tested the potential of GRN510 to limit fibrosis induced by bleomycin in mTERT heterozygous mice. Treatment with GRN510 at 10 mg/kg/day activated telomerase 2–4 fold both in hematopoietic progenitors ex vivo and in bone marrow and lung tissue in vivo, respectively. Telomerase activation was countered by co-treatment with Imetelstat (GRN163L), a potent telomerase inhibitor. In this model of bleomycin-induced fibrosis, treatment with GRN510 suppressed the development of fibrosis and accumulation of senescent cells in the lung via a mechanism dependent upon telomerase activation. Treatment of small airway epithelial cells (SAEC) or lung fibroblasts ex vivo with GRN510 revealed telomerase activating and replicative lifespan promoting effects only in the SAEC, suggesting that the mechanism accounting for the protective effects of GRN510 against induced lung fibrosis involves specific types of lung cells. Together, these results support the use of small molecule activators of telomerase in therapies to treat idiopathic pulmonary fibrosis.

The mouse lysyl oxidase-like 2 gene (mLOXL2) maps to chromosome 14 and is highly expressed in skin, lung and thymus.

The predicted amino acid sequence derived from a mouse expressed sequence tag (EST) contig contained two domains that are highly conserved among members of the lysyl oxidase gene family: a copper binding-site with four histidines and a catalytic domain that includes a tryptophan residue. This new cDNA sequence showed the highest level of sequence homology with the human loxl2 cDNA and suggested that it encoded the mouse equivalent of hLOXL2. The mLOXL2 gene was mapped to chromosome 14 by radiation hybrid analysis. The mLOXL2 locus was tightly linked with a LOD score over 9 to the marker D14Mit32. The mLOXL2 gene is expressed as a 4-kb mRNA in almost all tissues analyzed, with highest levels of mRNA in skin, lung and thymus.

Women and Lung Disease. Sex Differences and Global Health Disparities

There is growing evidence that a number of pulmonary diseases affect women differently and with a greater degree of severity than men. The causes for such sex disparity is the focus of this Blue Conference Perspective review, which explores basic cellular and molecular mechanisms, life stages, and clinical outcomes based on environmental, sociocultural, occupational, and infectious scenarios, as well as medical health beliefs. Owing to the breadth of issues related to women and lung disease, we present examples of both basic and clinical concepts that may be the cause for pulmonary disease disparity in women. These examples include those diseases that predominantly affect women, as well as the rising incidence among women for diseases traditionally occurring in men, such as chronic obstructive pulmonary disease. Sociocultural implications of pulmonary disease attributable to biomass burning and infectious diseases among women in low- to middle-income countries are reviewed, as are disparities in respiratory health among sexual minority women in high-income countries. The implications of the use of complementary and alternative medicine by women to influence respiratory disease are examined, and future directions for research on women and respiratory health are provided.

Senescent Cells Contribute to the Physiological Remodeling of Aged Lungs

Age-associated decline in organ function governs life span. We determined the effect of aging on lung function and cellular/molecular changes of 8- to 32-month old mice. Proteomic analysis of lung matrix indicated significant compositional changes with advanced age consistent with a profibrotic environment that leads to a significant increase in dynamic compliance and airway resistance. The excess of matrix proteins deposition was associated modestly with the activation of myofibroblasts and transforming growth factor-beta signaling pathway. More importantly, detection of senescent cells in the lungs increased with age and these cells contributed toward the excess extracellular matrix deposition observed in our aged mouse model and in elderly human samples. Mechanistic target of rapamycin (mTOR)/AKT activity was enhanced in aged mouse lungs compared with those from younger mice associated with the increased expression of the histone variant protein, MH2A, a marker for aging and potentially for senescence. Introduction in the mouse diet of rapamycin, significantly blocked the mTOR activity and limited the activation of myofibroblasts but did not result in a reduction in lung collagen deposition unless it was associated with prevention of cellular senescence. Together these data indicate that cellular senescence significantly contributes to the extracellular matrix changes associated with aging in a mTOR 1-dependent mechanism.