ADAMTS5 Deficiency Protects Mice From Chronic Tobacco Smoking-induced Intervertebral Disc Degeneration.

STUDY DESIGN: ADAMTS5-deficient and wild type (WT) mice were chronically exposed to tobacco smoke to investigate effects on intervertebral disc degeneration (IDD). OBJECTIVE: The aim of this study was to demonstrate a role for ADAMTS5 in mediating tobacco smoking-induced IDD. SUMMARY OF BACKGROUND DATA: We previously demonstrated that chronic tobacco smoking causes IDD in mice because, in part, of proteolytic destruction of disc aggrecan. However, it was unknown which matrix proteinase(s) drive these detrimental effects. METHODS: Three-month-old WT (C57BL/6) and ADAMTS5 mice were chronically exposed to tobacco smoke (four cigarettes/day, 5 day/week for 6 months). ADAMTS-mediated cleavage of disc aggrecan was analyzed by Western blot. Disc total glycosaminoglycan (GAG) content was assessed by dimethyl methylene blue assay and safranin O/fast green histology. Vertebral osteoporosity was measured by microcomputed tomography. Human nucleus pulposus (hNP) cell cultures were also exposed directly to tobacco smoke extract (TSE), a condensate containing the water-soluble compounds inhaled by smokers, to measure ADAMTS5 expression and ADAMTS-mediated cleavage of aggrecan. Activation of nuclear factor (NF)-κB, a family of transcription factors essential for modulating the cellular response to stress, was measured by immunofluorescence assay. RESULTS: Genetic depletion of ADAMTS5 prevented vertebral bone loss, substantially reduced loss of disc GAG content, and completely obviated ADAMTS-mediated proteolysis of disc aggrecan within its interglobular domain (IGD) in mice following exposure to tobacco smoke. hNP cell cultures exposed to TSE also resulted in upregulation of ADAMTS5 protein expression and a concomitant increase in ADAMTS-mediated cleavage within aggrecan IGD. Activation of NF-κB, known to be required for ADAMTS5 gene expression, was observed in both TSE-treated hNP cell cultures and disc tissue of tobacco smoke-exposed mice. CONCLUSION: The findings demonstrate that ADAMTS5 is the primary aggrecanase mediating smoking-induced disc aggrecanolysis and IDD. Mouse models of chronic tobacco smoking are important and useful for probing the mechanisms of disc aggrecan catabolism and IDD. LEVEL OF EVIDENCE: N/A.

Repression of CC16 by cigarette smoke (CS) exposure.

Club (Clara) Cell Secretory Protein (CCSP, or CC16) is produced mainly by non-ciliated airway epithelial cells including bronchiolar club cells and the change of its expression has been shown to associate with the progress and severity of Chronic Obstructive Pulmonary Disease (COPD). In an animal model, the lack of CC16 renders the animal susceptible to the tumorigenic effect of a major CS carcinogen. A recent population-based Tucson Epidemiological Study of Airway Obstructive Diseases (TESAOD) has indicated that the low serum CC16 concentration is closely linked with the smoke-related mortality, particularly that driven by the lung cancer. However, the study of CC16 expression in well-defined smoke exposure models has been lacking, and there is no experimental support for the potential causal link between CC16 and CS-induced pathophysiological changes in the lung. In the present study, we have found that airway CC16 expression was significantly repressed in COPD patients, in monkey CS exposure model, and in CS-induced mouse model of COPD. Additionally, the lack of CC16 exacerbated airway inflammation and alveolar loss in the mouse model. Therefore, CC16 may play an important protective role in CS-related diseases.

SPLUNC1 deficiency enhances airway eosinophilic inflammation in mice.

Short palate, lung and nasal epithelium clone 1 (SPLUNC1) is enriched in normal airway lining fluid, but is significantly reduced in airway epithelium exposed to a Th2 cytokine milieu. The role of SPLUNC1 in modulating airway allergic inflammation (e.g., eosinophils) remains unknown. We used SPLUNC1 knockout (KO) and littermate wild-type (C57BL/6 background) mice and recombinant SPLUNC1 protein to determine the impact of SPLUNC1 on airway allergic/eosinophilic inflammation, and to investigate the underlying mechanisms. An acute ovalbumin (OVA) sensitization and challenge protocol was used to induce murine airway allergic inflammation (e.g., eosinophils, eotaxin-2, and Th2 cytokines). Our results showed that SPLUNC1 in the bronchoalveolar lavage fluid of OVA-challenged wild-type mice was significantly reduced (P < 0.05), which was negatively correlated with levels of lung eosinophilic inflammation. Moreover, SPLUNC1 KO mice demonstrated significantly higher numbers of eosinophils in the lung after OVA challenges than did wild-type mice. Alveolar macrophages isolated from OVA-challenged SPLUNC1 KO versus wild-type mice had higher concentrations of baseline eotaxin-2 that was amplified by LPS (a known risk factor for exacerbating asthma). Human recombinant SPLUNC1 protein was applied to alveolar macrophages to study the regulation of eotaxin-2 in the context of Th2 cytokine and LPS stimulation. Recombinant SPLUNC1 protein attenuated LPS-induced eotaxin-2 production in Th2 cytokine-pretreated murine macrophages. These findings demonstrate that SPLUNC1 inhibits airway eosinophilic inflammation in allergic mice, in part by reducing eotaxin-2 production in alveolar macrophages.

Matrix metalloproteinase-14 mediates a phenotypic shift in the airways to increase mucin production.

RATIONALE: Induced mainly by cigarette smoking, chronic obstructive pulmonary disease (COPD) is a global public health problem characterized by progressive difficulty in breathing and increased mucin production. Previously, we reported that acrolein levels found in COPD sputum could activate matrix metalloproteinase-9 (MMP9). OBJECTIVES: To determine whether acrolein increases expression and activity of MMP14, a critical membrane-bound endopeptidase that can initial a MMP-activation cascade. METHODS: MMP14 activity and adduct formation were measured following direct acrolein treatment. MMP14 expression and activity was measured in human airway epithelial cells. MMP14 immunohistochemistry was performed with COPD tissue, and in acrolein- or tobacco-exposed mice. MEASUREMENTS AND MAIN RESULTS: In a cell-free system, acrolein, in concentrations equal to those found in COPD sputum, directly adducted cysteine 319 in the MMP14 hemopexin-like domain and activated MMP14. In cells, acrolein increased MMP14 activity, which was inhibited by a proprotein convertase inhibitor, hexa-d-arginine. In the airway epithelium of COPD subjects, immunoreactive MMP14 protein increased. In mouse lung, acrolein or tobacco smoke increased lung MMP14 activity and protein. In cells, acrolein-induced MMP14 transcripts were inhibited by an epidermal growth factor receptor (EGFR) neutralizing antibody, EGFR kinase inhibitor, metalloproteinase inhibitor, or mitogen-activated protein kinase (MAPK) 3/2 or MAPK8 inhibitors, but not a MAPK14 inhibitor. Decreasing the MMP14 protein and activity in vitro by small interfering (si)RNA to MMP14 diminished the acrolein-induced MUC5AC transcripts. In acrolein-exposed mice or transgenic mice with lung-specific transforming growth factor-alpha (an EGFR ligand) expression, lung MMP14 and MUC5AC levels increased and these effects were inhibited by a EGFR inhibitor, erlotinib. CONCLUSIONS: Taken together, these findings implicate acrolein-induced MMP14 expression and activity in mucin production in COPD.

Conditional stabilization of beta-catenin expands the pool of lung stem cells.

Maintenance of classic stem cell hierarchies is dependent upon stem cell self-renewal mediated in part by Wnt/beta-catenin regulation of the cell cycle. This function is critical in rapidly renewing tissues due to the obligate role played by the tissue stem cell. However, the stem cell hierarchy responsible for maintenance of the conducting airway epithelium is distinct from classic stem cell hierarchies. The epithelium of conducting airways is maintained by transit-amplifying cells in the steady state; rare bronchiolar stem cells are activated to participate in epithelial repair only following depletion of transit-amplifying cells. Here, we investigate how signaling through beta-catenin affects establishment and maintenance of the stem cell hierarchy within the slowly renewing epithelium of the lung. Conditional potentiation of beta-catenin signaling in the embryonic lung results in amplification of airway stem cells through attenuated differentiation rather than augmented proliferation. Our data demonstrate that the differentiation-modulating activities of stabilized beta-catenin account for expansion of tissue stem cells.

Cytochrome c acts as a cardiolipin oxygenase required for release of proapoptotic factors.

Programmed death (apoptosis) is turned on in damaged or unwanted cells to secure their clean and safe self-elimination. The initial apoptotic events are coordinated in mitochondria, whereby several proapoptotic factors, including cytochrome c, are released into the cytosol to trigger caspase cascades. The release mechanisms include interactions of B-cell/lymphoma 2 family proteins with a mitochondria-specific phospholipid, cardiolipin, to cause permeabilization of the outer mitochondrial membrane. Using oxidative lipidomics, we showed that cardiolipin is the only phospholipid in mitochondria that undergoes early oxidation during apoptosis. The oxidation is catalyzed by a cardiolipin-specific peroxidase activity of cardiolipin-bound cytochrome c. In a previously undescribed step in apoptosis, we showed that oxidized cardiolipin is required for the release of proapoptotic factors. These results provide insight into the role of reactive oxygen species in triggering the cell-death pathway and describe an early role for cytochrome c before caspase activation.

Identification of a novel MAGE D2 antisense RNA transcript in human tissues.

Using cDNA microarray analysis, we identified a cDNA clone, DD74, from primary human bronchial epithelial cells, which exhibits increased expression in vitro after treatment with all-trans retinoic acid. This clone corresponded to MAGE D2 mRNA, a gene previously identified to be upregulated in several cancer tissues. Surprisingly, in situ hybridization of lung tissue demonstrated positive hybridization signals with sense, but not antisense, MAGE D2-specific cRNA probes. Examination of several cell lines by Northern blot hybridization confirmed significant expression of two RNA bands. With strand-specific riboprobes, we identified a 2.0kb RNA transcript with the antisense probe as expected and identified a 4.1kb transcript by the sense probe. Further sequence analysis of the 4.1kb transcript revealed at least a 509 nucleotide sequence exactly complementary to the 2.0kb MAGE D2 mRNA sequence. This MAGE D2i sequence contains unique structural features not shared with those of previously described antisense transcripts. Identification of this transcript potentially has important implications for future studies examining MAGE D2 expression patterns in cancer and normal tissues.

Phosphorylation of tumor necrosis factor receptor 1 (p55) protects macrophages from silica-induced apoptosis.

Macrophages play a fundamental role in silicosis in part by removing silica particles and producing inflammatory mediators in response to silica. Tumor necrosis factor alpha (TNFalpha) is a prominent mediator in silicosis. Silica induction of apoptosis in macrophages might be mediated by TNFalpha. However, TNFalpha also activates signal transduction pathways (NF-kappaB and AP-1) that rescue cells from apoptosis. Therefore, we studied the TNFalpha-mediated mechanisms that confer macrophage protection against the pro-apoptotic effects of silica. We will show that exposure to silica induced TNFalpha production by RAW 264.7 cells, but not by IC-21. Silica-induced activation of NF-kappaB and AP-1 was only observed in RAW 264.7 macrophages. ERK activation in response to silica exposure was only observed in RAW 264.7 macrophages, whereas activation of p38 phosphorylation was predominantly observed in IC-21 macrophages. No changes in JNK activity were observed in either cell line in response to silica exposure. Silica induced apoptosis in both macrophage cell lines, but the induction of apoptosis was significantly larger in IC-21 cells. Protection against apoptosis in RAW 264.7 cells in response to silica was mediated by enhanced NF-kappaB activation and ERK-mediated phosphorylation of the p55 TNFalpha receptor. Inhibition of these two protective mechanisms by specific pharmacological inhibitors or transfection of dominant negative mutants that inhibit IkappaBalpha or ERK phosphorylation significantly increased silica-induced apoptosis in RAW 264.7 macrophages. These data suggest that NF-kappaB activation and ERK-mediated phosphorylation of the p55 TNF receptor are important cell survival mechanisms in the macrophage response to silica exposure.