FBXL19 Targeted STK11 Degradation Enhances Cigarette Smoke-Induced Airway Epithelial Cell Cytotoxicity.

Objective: To investigate the effects of cigarette smoke (CS) on Serine/Threonine Kinase 11 (STK11) and to determine STK11's role in CS-induced airway epithelial cell cytotoxicity.Methods: STK11 expression levels in the lung tissues of smokers with or without COPD and mice exposed to CS or room air (RA) were determined by immunoblotting and RT-PCR. BEAS-2Bs-human bronchial airway epithelial cells were exposed to CS extract (CSE), and the changes in STK11 expression levels were determined by immunoblotting and RT-PCR. BEAS-2B cells were transfected with STK11-specific siRNA or STK11 expression plasmid, and the effects of CSE on airway epithelial cell cytotoxicity were measured. To determine the specific STK11 degradation-proteolytic pathway, BEAS-2Bs were treated with cycloheximide alone or combined with MG132 or leupeptin. Finally, to identify the F-box protein mediating the STK11 degradation, a screening assay was performed using transfection with a panel of FBXL E3 ligase subunits.Results: STK11 protein levels were significantly decreased in the lung tissues of smokers with COPD relative to smokers without COPD. STK11 protein levels were also significantly decreased in mouse lung tissues exposed to CS compared to RA. Exposure to CSE shortened the STK11 mRNA and protein half-life to 4 h in BEAS-2B cells. STK11 protein overexpression attenuated the CSE-induced cytotoxicity; in contrast, its knockdown augmented CSE-induced cytotoxicity. FBXL19 mediates CSE-induced STK11 protein degradation via the ubiquitin-proteasome pathway in cultured BEAS-2B cells. FBXL19 overexpression led to accelerated STK11 ubiquitination and degradation in a dose-dependent manner.Conclusions: Our results suggest that CSE enhances the degradation of STK11 protein in airway epithelial cells via the FBXL19-mediated ubiquitin-proteasomal pathway, leading to augmented cell death.HIGHLIGHTSLung tissues of COPD-smokers exhibited a decreased STK11 RNA and protein expression.STK11 overexpression attenuates CS-induced airway epithelial cell cytotoxicity.STK11 depletion augments CS-induced airway epithelial cell cytotoxicity.CS diminishes STK11 via FBXL19-mediated ubiquitin-proteasome degradation.

SARS-CoV-2 Accessory Protein Orf7b Induces Lung Injury via c-Myc Mediated Apoptosis and Ferroptosis.

The pandemic of coronavirus disease 2019 (COVID-19) has been the foremost modern global public health challenge. The airway is the primary target in severe acute respiratory distress syndrome coronavirus 2 (SARS-CoV-2) infection, with substantial cell death and lung injury being signature hallmarks of exposure. The viral factors that contribute to cell death and lung injury remain incompletely understood. Thus, this study investigated the role of open reading frame 7b (Orf7b), an accessory protein of the virus, in causing lung injury. In screening viral proteins, we identified Orf7b as one of the major viral factors that mediates lung epithelial cell death. Overexpression of Orf7b leads to apoptosis and ferroptosis in lung epithelial cells, and inhibitors of apoptosis and ferroptosis ablate Orf7b-induced cell death. Orf7b upregulates the transcription regulator, c-Myc, which is integral in the activation of lung cell death pathways. Depletion of c-Myc alleviates both apoptotic and ferroptotic cell deaths and lung injury in mouse models. Our study suggests a major role of Orf7b in the cell death and lung injury attributable to COVID-19 exposure, supporting it as a potential therapeutic target.

Cigarette smoking is a secondary cause of folliculin loss.

BACKGROUND: Birt-Hogg-Dubé syndrome (BHD) is a clinical syndrome manifesting with cystic lung disease and pneumothorax. Features of BHD result from the loss-of-function mutations of the folliculin (FLCN) gene. Chronic obstructive pulmonary disease (COPD), characterised by an irreversible airflow limitation, is primarily caused by cigarette smoking. OBJECTIVE: Given that COPD often shares structural features with BHD, we investigated the link between COPD, cigarette smoke (CS) exposure and FLCN expression. METHODS: We measured the expression of FLCN in human COPD lungs and CS-exposed mouse lungs, as well as in CS extract (CSE)-exposed immortalised human airway epithelial cells by immunoblotting. RESULTS: We found that the lung FLCN protein levels in smokers with COPD and CS exposure mice exhibit a marked decrease compared with smokers without COPD and room air exposure mice, respectively. We confirmed CS induced degradation of FLCN in immortalised human bronchial epithelial Beas-2B cells via ubiquitin proteasome system. Further, siRNA targeting FLCN enhanced CSE-induced cytotoxicity. By contrast, FLCN overexpression protected cells from CSE-induced cytotoxicity. We found that FBXO23, the ubiquitin E3 ligase subunit, specifically binds to and targets FLCN for degradation. Inhibition of ATM (ataxia-telangiectasia mutated) attenuated CSE induced FLCN degradation, suggesting a role of ATM in FLCN proteolysis. We further confirmed that the mutant of major FLCN phosphorylation site serine 62A is resistant to CSE-induced degradation and cytotoxicity. CONCLUSIONS: Our study demonstrates that CS exposure is a secondary cause of FLCN deficiency due to the enhanced proteolysis, which promoted airway epithelial cell death.

Protein arginine N-methyltransferase 4 (PRMT4) contributes to lymphopenia in experimental sepsis.

BACKGROUND: One hallmark of sepsis is the reduced number of lymphocytes, termed lymphopenia, that occurs from decreased lymphocyte proliferation or increased cell death contributing to immune suppression. Histone modification enzymes regulate immunity by their epigenetic and non-epigenetic functions; however, the role of these enzymes in lymphopenia remains elusive. METHODS: We used molecular biological approaches to investigate the high expression and function of a chromatin modulator protein arginine N-methyltransferase 4 (PRMT4)/coactivator-associated arginine methyltransferase 1 in human samples from septic patients and cellular and animal septic models. RESULTS: We identified that PRMT4 is elevated systemically in septic patients and experimental sepsis. Gram-negative bacteria and their derived endotoxin lipopolysaccharide (LPS) increased PRMT4 in B and T lymphocytes and THP-1 monocytes. Single-cell RNA sequencing results indicate an increase of PRMT4 gene expression in activated T lymphocytes. Augmented PRMT4 is crucial for inducing lymphocyte apoptosis but not monocyte THP-1 cells. Ectopic expression of PRMT4 protein caused substantial lymphocyte death via caspase 3-mediated cell death signalling, and knockout of PRMT4 abolished LPS-mediated lymphocyte death. PRMT4 inhibition with a small molecule compound attenuated lymphocyte death in complementary models of sepsis. CONCLUSIONS: These findings demonstrate a previously uncharacterised role of a key chromatin modulator in lymphocyte survival that may shed light on devising therapeutic modalities to lessen the severity of septic immunosuppression.

Stability of SARS-CoV-2-Encoded Proteins and Their Antibody Levels Correlate with Interleukin 6 in COVID-19 Patients.

The spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has become a severe global public health crisis. Therefore, understanding the molecular details of SARS-CoV-2 will be critical for fighting the virus's spread and preventing future pandemics. In this study, we globally profiled the stability of SARS-CoV-2-encoded proteins, studied their degradation pathways, and determined their correlation with the antibody responses in patient plasma. We identified 18 proteins with unstable half-lives and 6 relatively stable proteins with longer half-lives. The labile SARS-CoV-2 proteins were degraded mainly by the ubiquitin-proteasome pathway. We also observed a significant correlation between antibody levels and protein half-lives, which indicated that a stable antigen of SARS-CoV-2 could be more effective for eliciting antibody responses. In addition, levels of antiviral antibodies targeting NSP10 were found to be negatively correlated with systemic levels of interleukin 6 (IL-6) in patients. These findings may facilitate the development of novel therapeutic or diagnostic approaches. IMPORTANCE SARS-CoV-2, the etiological cause of COVID-19, carries 29 genes in its genome. However, our knowledge of the viral proteins in biological and biochemical aspects is limited. In this study, we globally profiled the stability of the viral proteins in living lung epithelial cells. Importantly, the labile SARS-CoV-2-encoded proteins were mainly degraded through the ubiquitin-proteasome pathway. Stable proteins, including spike and nucleocapsid, of SARS-CoV-2 were more effective in eliciting antibody production. The levels of antiviral antibodies targeting NSP10 were negatively correlated with systemic levels of IL-6 in COVID-19 patients.

Endotoxin stabilizes protein arginine methyltransferase 4 (PRMT4) protein triggering death of lung epithelia.

Lung epithelial cell death is a prominent feature of acute lung injury and acute respiratory distress syndrome (ALI/ARDS), which results from severe pulmonary infection leading to respiratory failure. Multiple mechanisms are believed to contribute to the death of epithelia; however, limited data propose a role for epigenetic modifiers. In this study, we report that a chromatin modulator protein arginine N-methyltransferase 4/coactivator-associated arginine methyltransferase 1 (PRMT4/CARM1) is elevated in human lung tissues with pneumonia and in experimental lung injury models. Here PRMT4 is normally targeted for its degradation by an E3 ubiquitin ligase, SCFFBXO9, that interacts with PRMT4 via a phosphodegron to ubiquitinate the chromatin modulator at K228 leading to its proteasomal degradation. Bacterial-derived endotoxin reduced levels of SCFFBXO9 thus increasing PRMT4 cellular concentrations linked to epithelial cell death. Elevated PRMT4 protein caused substantial epithelial cell death via caspase 3-mediated cell death signaling, and depletion of PRMT4 abolished LPS-mediated epithelial cell death both in cellular and murine injury models. These findings implicate a unique molecular interaction between SCFFBXO9 and PRMT4 and its regulation by endotoxin that impacts the life span of lung epithelia, which may play a key role in the pathobiology of tissue injury observed during critical respiratory illness.

Thrombospondin-1 Restricts Interleukin-36γ-Mediated Neutrophilic Inflammation during Pseudomonas aeruginosa Pulmonary Infection.

Interleukin-36γ (IL-36γ), a member of the IL-1 cytokine superfamily, amplifies lung inflammation and impairs host defense during acute pulmonary Pseudomonas aeruginosa infection. To be fully active, IL-36γ is cleaved at its N-terminal region by proteases such as neutrophil elastase (NE) and cathepsin S (CatS). However, it remains unclear whether limiting extracellular proteolysis restrains the inflammatory cascade triggered by IL-36γ during P. aeruginosa infection. Thrombospondin-1 (TSP-1) is a matricellular protein with inhibitory activity against NE and the pathogen-secreted Pseudomonas elastase LasB-both proteases implicated in amplifying inflammation. We hypothesized that TSP-1 tempers the inflammatory response during lung P. aeruginosa infection by inhibiting the proteolytic environment required for IL-36γ activation. Compared to wild-type (WT) mice, TSP-1-deficient (Thbs1-/-) mice exhibited a hyperinflammatory response in the lungs during P. aeruginosa infection, with increased cytokine production and an unrestrained extracellular proteolytic environment characterized by higher free NE and LasB, but not CatS activity. LasB cleaved IL-36γ proximally to M19 at a cleavage site distinct from those generated by NE and CatS, which cleave IL-36γ proximally to Y16 and S18, respectively. N-terminal truncation experiments in silico predicted that the M19 and the S18 isoforms bind the IL-36R complex almost identically. IL-36γ neutralization ameliorated the hyperinflammatory response and improved lung immunity in Thbs1-/- mice during P. aeruginosa infection. Moreover, administration of cleaved IL-36γ induced cytokine production and neutrophil recruitment and activation that was accentuated in Thbs1-/- mice lungs. Collectively, our data show that TSP-1 regulates lung neutrophilic inflammation and facilitates host defense by restraining the extracellular proteolytic environment required for IL-36γ activation.IMPORTANCEPseudomonas aeruginosa pulmonary infection can lead to exaggerated neutrophilic inflammation and tissue destruction, yet host factors that regulate the neutrophilic response are not fully known. IL-36γ is a proinflammatory cytokine that dramatically increases in bioactivity following N-terminal processing by proteases. Here, we demonstrate that thrombospondin-1, a host matricellular protein, limits N-terminal processing of IL-36γ by neutrophil elastase and the Pseudomonas aeruginosa-secreted protease LasB. Thrombospondin-1-deficient mice (Thbs1-/-) exhibit a hyperinflammatory response following infection. Whereas IL-36γ neutralization reduces inflammatory cytokine production, limits neutrophil activation, and improves host defense in Thbs1-/- mice, cleaved IL-36γ administration amplifies neutrophilic inflammation in Thbs1-/- mice. Our findings indicate that thrombospondin-1 guards against feed-forward neutrophilic inflammation mediated by IL-36γ in the lung by restraining the extracellular proteolytic environment.

Single cell RNA sequencing identifies IGFBP5 and QKI as ciliated epithelial cell genes associated with severe COPD.

BACKGROUND: Whole lung tissue transcriptomic profiling studies in chronic obstructive pulmonary disease (COPD) have led to the identification of several genes associated with the severity of airflow limitation and/or the presence of emphysema, however, the cell types driving these gene expression signatures remain unidentified. METHODS: To determine cell specific transcriptomic changes in severe COPD, we conducted single-cell RNA sequencing (scRNA seq) on n = 29,961 cells from the peripheral lung parenchymal tissue of nonsmoking subjects without underlying lung disease (n = 3) and patients with severe COPD (n = 3). The cell type composition and cell specific gene expression signature was assessed. Gene set enrichment analysis (GSEA) was used to identify the specific cell types contributing to the previously reported transcriptomic signatures. RESULTS: T-distributed stochastic neighbor embedding and clustering of scRNA seq data revealed a total of 17 distinct populations. Among them, the populations with more differentially expressed genes in cases vs. controls (log fold change >|0.4| and FDR = 0.05) were: monocytes (n = 1499); macrophages (n = 868) and ciliated epithelial cells (n = 590), respectively. Using GSEA, we found that only ciliated and cytotoxic T cells manifested a trend towards enrichment of the previously reported 127 regional emphysema gene signatures (normalized enrichment score [NES] = 1.28 and = 1.33, FDR = 0.085 and = 0.092 respectively). Among the significantly altered genes present in ciliated epithelial cells of the COPD lungs, QKI and IGFBP5 protein levels were also found to be altered in the COPD lungs. CONCLUSIONS: scRNA seq is useful for identifying transcriptional changes and possibly individual protein levels that may contribute to the development of emphysema in a cell-type specific manner.

Cigarette smoke extract induces airway epithelial cell death via repressing PRMT6/AKT signaling.

Chronic obstructive pulmonary disease (COPD) is a severe public health threat world-wide. Cigarette smoke (CS)-induced airway epithelial cell death is a major pathway of pathogenesis in emphysema, a subtype of COPD. Protein arginine methyltransferase 6 (PRMT6) is a type I PRMT that catalyzes mono- and di-methylation on arginine residues within histone and non-histone proteins to modulate a variety of life processes, such as apoptosis. However, its role in CS-induced lung epithelial death has not been fully elucidated. Here we report that PRMT6 was decreased in mouse lung tissues from a cigarette smoke extract (CSE)-mediated experimental emphysematous model and in CSE treated or cigarette smoke exposed lung epithelial cells. Depletion of PRMT6 increased the protein levels of phosphatase PTEN and PI3K regulatory subunit p85 but decreased a downstream kinase PDK1, resulting in AKT dephosphorylation and thereafter, lung epithelial cell death. Knockout of PRMT6 inhibited epithelial survival and promoted CSE-mediated epithelial cell death, while ectopic expression of PRMT6 protein partially reversed epithelial cell death via PI3K/AKT-mediated cell survival signaling in CSE cellular models. These findings demonstrate that PRMT6 plays a crucial role in CS-induced bronchial epithelial cell death that may be a potential therapeutic target against the airway cell death in CS-induced COPD.

Pseudomonas Aeruginosa Induced Cell Death in Acute Lung Injury and Acute Respiratory Distress Syndrome.

Pseudomonas aeruginosa is an important opportunistic pathogen responsible for the cause of acute lung injury and acute respiratory distress syndrome. P. aeruginosa isthe leading species isolated from patients with nosocomial infection and is detected in almost all the patients with long term ventilation in critical care units. P. aeruginosa infection is also the leading cause of deleterious chronic lung infections in patients suffering from cystic fibrosis as well as the major reason for morbidity in people with chronic obstructive pulmonary disease. P. aeruginosa infections are linked to diseases with high mortality rates and are challenging for treatment, for which no effective remedies have been developed. Massive lung epithelial cell death is a hallmark of severe acute lung injury and acute respiratory distress syndrome caused by P. aeruginosa infection. Lung epithelial cell death poses serious challenges to air barrier and structural integrity that may lead to edema, cytokine secretion, inflammatory infiltration, and hypoxia. Here we review different types of cell death caused by P. aeruginosa serving as a starting point for the diseases it is responsible for causing. We also review the different mechanisms of cell death and potential therapeutics in countering the serious challenges presented by this deadly bacterium.

The Role of Deubiquitinating Enzymes in Acute Lung Injury and Acute Respiratory Distress Syndrome.

Acute lung injury and acute respiratory distress syndrome (ALI/ARDS) are characterized by an inflammatory response, alveolar edema, and hypoxemia. ARDS occurs most often in the settings of pneumonia, sepsis, aspiration of gastric contents, or severe trauma. The prevalence of ARDS is approximately 10% in patients of intensive care. There is no effective remedy with mortality high at 30-40%. Most functional proteins are dynamic and stringently governed by ubiquitin proteasomal degradation. Protein ubiquitination is reversible, the covalently attached monoubiquitin or polyubiquitin moieties within the targeted protein can be removed by a group of enzymes called deubiquitinating enzymes (DUBs). Deubiquitination plays an important role in the pathobiology of ALI/ARDS as it regulates proteins critical in engagement of the alveolo-capillary barrier and in the inflammatory response. In this review, we provide an overview of how DUBs emerge in pathogen-induced pulmonary inflammation and related aspects in ALI/ARDS. Better understanding of deubiquitination-relatedsignaling may lead to novel therapeutic approaches by targeting specific elements of the deubiquitination pathways.

Studying Effects of Cigarette Smoke on Pseudomonas Infection in Lung Epithelial Cells.

Cigarette smoking is the major etiological cause for lung emphysema and chronic obstructive pulmonary disease (COPD). Cigarette smoking also promotes susceptibility to bacterial infections in the respiratory system. However, the effects of cigarette smoking on bacterial infections in human lung epithelial cells have yet to be thoroughly studied. Described here is a detailed protocol for the preparation of cigarette smoking extracts (CSE), treatment of human lung epithelial cells with CSE, and bacterial infection and infection determination. CSE was prepared with a conventional method. Lung epithelial cells were treated with 4% CSE for 3 h. CSE-treated cells were, then, infected with Pseudomonas at a multiplicity of infection (MOI) of 10. Bacterial loads of the cells were determined by three different methods. The results showed that CSE increased Pseudomonas load in lung epithelial cells. This protocol, therefore, provides a simple and reproducible approach to study the effect of cigarette smoke on bacterial infections in lung epithelial cells.

Tumor Necrosis Factor Alpha Regulates Skeletal Myogenesis by Inhibiting SP1 Interaction with cis-Acting Regulatory Elements within the Fbxl2 Gene Promoter.

FBXL2 is an important ubiquitin E3 ligase component that modulates inflammatory signaling and cell cycle progression, but its molecular regulation is largely unknown. Here, we show that tumor necrosis factor alpha (TNF-α), a critical cytokine linked to the inflammatory response during skeletal muscle regeneration, suppressed Fbxl2 mRNA expression in C2C12 myoblasts and triggered significant alterations in cell cycle, metabolic, and protein translation processes. Gene silencing of Fbxl2 in skeletal myoblasts resulted in increased proliferative responses characterized by activation of mitogen-activated protein (MAP) kinases and nuclear factor kappa B and decreased myogenic differentiation, as reflected by reduced expression of myogenin and impaired myotube formation. TNF-α did not destabilize the Fbxl2 transcript (half-life [t1/2], ∼10 h) but inhibited SP1 transactivation of its core promoter, localized to bp -160 to +42 within the proximal 5' flanking region of the Fbxl2 gene. Chromatin immunoprecipitation and gel shift studies indicated that SP1 interacted with the Fbxl2 promoter during cellular differentiation, an effect that was less pronounced during proliferation or after TNF-α exposure. TNF-α, via activation of JNK, mediated phosphorylation of SP1 that impaired its binding to the Fbxl2 promoter, resulting in reduced transcriptional activity. The results suggest that SP1 transcriptional activation of Fbxl2 is required for skeletal muscle differentiation, a process that is interrupted by a key proinflammatory myopathic cytokine.IMPORTANCE Skeletal muscle regeneration and repair involve the recruitment and proliferation of resident satellite cells that exit the cell cycle during the process of myogenic differentiation to form myofibers. We demonstrate that the ubiquitin E3 ligase subunit FBXL2 is essential for skeletal myogenesis through its important effects on cell cycle progression and cell proliferative signaling. Further, we characterize a new mechanism whereby sustained stimulation by a major proinflammatory cytokine, TNF-α, regulates skeletal myogenesis by inhibiting the interaction of SP1 with the Fbxl2 core promoter in proliferating myoblasts. Our findings contribute to the understanding of skeletal muscle regeneration through the identification of Fbxl2 as both a critical regulator of myogenic proliferative processes and a susceptible gene target during inflammatory stimulation by TNF-α in skeletal muscle. Modulation of Fbxl2 activity may have relevance to disorders of muscle wasting associated with sustained proinflammatory signaling.

Cigarette smoke exposure enhances transforming acidic coiled-coil-containing protein 2 turnover and thereby promotes emphysema.

Our integrative genomic and functional analysis identified transforming acidic coiled-coil-containing protein 2 (TACC2) as a chronic obstructive pulmonary disease (COPD) candidate gene. Here, we found that smokers with COPD exhibit a marked decrease in lung TACC2 protein levels relative to smokers without COPD. Single cell RNA sequencing reveals that TACC2 is expressed primarily in lung epithelial cells in normal human lungs. Furthermore, suppression of TACC2 expression impairs the efficiency of homologous recombination repair and augments spontaneous and cigarette smoke extract-induced (CSE-induced) DNA damage and cytotoxicity in immortalized human bronchial epithelial cells. By contrast, enforced expression of TACC2 attenuates the CSE effects. We also found that CSE enhances TACC2 degradation via the ubiquitin-proteasome system mediated by the ubiquitin E3 ligase subunit, F box L7. Furthermore, cellularly expressed TACC2 proteins harboring naturally occurring mutations exhibited altered protein lifespan coupled with modified DNA damage repair and cytotoxic responses. CS triggers emphysematous changes accompanied by accumulated DNA damage, apoptosis of alveolar epithelia, and lung inflammation in Tacc2-/- compared with Tacc2+/+ mice. Our results suggest that CS destabilizes TACC2 protein in lung epithelia by the ubiquitin proteasome system, leading to subsequent DNA damage, cytotoxicity, and emphysema.

Cigarette smoke extract modulates Pseudomonas aeruginosa bacterial load via USP25/HDAC11 axis in lung epithelial cells.

Cigarette smoking increases susceptibility for microbial infection in respiratory system. However, the underlying molecular mechanism(s) is not fully elucidated. Here we report that cigarette smoking extract (CSE) increases bacterial load in lung epithelial cells via downregulation of the ubiquitin-specific protease 25 (USP25)/histone deacetylase 11 (HDAC11) axis. CSE treatment decreases HDAC11 at protein level in lung epithelial cells without significant changes of its transcription. Concomitantly, CSE treatment accelerates a ubiquitin-specific protease USP25 ubiquitination and degradation. Coimmunoprecipitation studies showed that USP25 associated with HDAC11. USP25 catalyzes deubiquitination of HDAC11, which regulates HDAC11 protein stability. CSE-mediated degradation of USP25 thereafter reduces HDAC11 at the protein level. Interestingly, CSE-downregulated USP25/HDAC11 axis increases the bacterial load of Pseudomonas aeruginosa in lung epithelial cells. These findings suggest that CSE-downregulated USP25 and HDAC11 may contribute to high susceptibility of bacterial infection in the cigarette smoking population.

RNA sequencing identifies common pathways between cigarette smoke exposure and replicative senescence in human airway epithelia.

BACKGROUND: Aging is affected by genetic and environmental factors, and cigarette smoking is strongly associated with accumulation of senescent cells. In this study, we wanted to identify genes that may potentially be beneficial for cell survival in response to cigarette smoke and thereby may contribute to development of cellular senescence. RESULTS: Primary human bronchial epithelial cells from five healthy donors were cultured, treated with or without 1.5% cigarette smoke extract (CSE) for 24 h or were passaged into replicative senescence. Transcriptome changes were monitored using RNA-seq in CSE and non-CSE exposed cells and those passaged into replicative senescence. We found that, among 1534 genes differentially regulated during senescence and 599 after CSE exposure, 243 were altered in both conditions, representing strong enrichment. Pathways and gene sets overrepresented in both conditions belonged to cellular processes that regulate reactive oxygen species, proteasome degradation, and NF-κB signaling. CONCLUSIONS: Our results offer insights into gene expression responses during cellular aging and cigarette smoke exposure, and identify potential molecular pathways that are altered by cigarette smoke and may also promote airway epithelial cell senescence.

Polygonum aviculare L. extract and quercetin attenuate contraction in airway smooth muscle.

Because of the serious side effects of the currently used bronchodilators, new compounds with similar functions must be developed. We screened several herbs and found that Polygonum aviculare L. contains ingredients that inhibit the precontraction of mouse and human airway smooth muscle (ASM). High K+-induced precontraction in ASM was completely inhibited by nifedipine, a selective blocker of L-type voltage-dependent Ca2+ channels (LVDCCs). However, nifedipine only partially reduced the precontraction induced by acetylcholine chloride (ACH). Additionally, the ACH-induced precontraction was partly reduced by pyrazole-3 (Pyr3), a selective blocker of TRPC3 and stromal interaction molecule (STIM)/Orai channels. These channel-mediated currents were inhibited by the compounds present in P. aviculare extracts, suggesting that this inhibition was mediated by LVDCCs, TRPC3 and/or STIM/Orai channels. Moreover, these channel-mediated currents were inhibited by quercetin, which is present in P. aviculare extracts. Furthermore, quercetin inhibited ACH-induced precontraction in ASM. Overall, our data indicate that the ethyl acetate fraction of P. aviculare and quercetin can inhibit Ca2+-permeant LVDCCs, TRPC3 and STIM/Orai channels, which inhibits the precontraction of ASM. These findings suggest that P. aviculare could be used to develop new bronchodilators to treat obstructive lung diseases such as asthma and chronic obstructive pulmonary disease.

LPS promotes HBO1 stability via USP25 to modulate inflammatory gene transcription in THP-1 cells.

The histone acetyltransferase HBO1 (Histone acetyltransferase binding to origin recognition complex 1, Myst2/Kat7) participates in a range of life processes including DNA replication and tumorigenesis. Recent studies revealed that HBO1 is involved in gene transcriptional activation. However, the molecular behavior of HBO1 in inflammation is yet to be studied. Here we report that endotoxin lipopolysaccharide (LPS) elevates HBO1 protein level via up-regulating UPS25 (ubiquitin specific peptidase 25) and alters inflammatory gene transcription in THP-1 monocytes and in human primary macrophages. LPS protects HBO1 from ubiquitin proteasomal degradation without significantly altering its transcription. By immunoprecipitation, we identified that HBO1 associates with a deubiquitinating enzyme USP25 in THP-1 cells. LPS increases protein level of USP25 resulting in accumulation of HBO1 by suppression of HBO1 ubiquitination. Stabilized-HBO1 modulates inflammatory gene transcription in THP-1 cells. These findings indicate that USP25 promotes stability of HBO1 in bacterial infection thereby enhances HBO1-mediated inflammatory gene transcription.

Distinct Effects of Ca2+ Sparks on Cerebral Artery and Airway Smooth Muscle Cell Tone in Mice and Humans.

The effects of Ca2+ sparks on cerebral artery smooth muscle cells (CASMCs) and airway smooth muscle cells (ASMCs) tone, as well as the underlying mechanisms, are not clear. In this investigation, we elucidated the underlying mechanisms of the distinct effects of Ca2+ sparks on cerebral artery smooth muscle cells (CASMCs) and airway smooth muscle cells (ASMCs) tone. In CASMCs, owing to the functional loss of Ca2+-activated Cl- (Clca) channels, Ca2+ sparks activated large-conductance Ca2+-activated K+ channels (BKs), resulting in a decreases in tone against a spontaneous depolarization-caused high tone in the resting state. In ASMCs, Ca2+ sparks induced relaxation through BKs and contraction via Clca channels. However, the integrated result was contraction because Ca2+ sparks activated BKs prior to Clca channels and Clca channels-induced depolarization was larger than BKs-caused hyperpolarization. However, the effects of Ca2+ sparks on both cell types were determined by L-type voltage-dependent Ca2+ channels (LVDCCs). In addition, compared with ASMCs, CASMCs had great and higher amplitude Ca2+ sparks, a higher density of BKs, and higher Ca2+ and voltage sensitivity of BKs. These differences enhanced the ability of Ca2+ sparks to decrease CASMC and to increase ASMC tone. The higher Ca2+ and voltage sensitivity of BKs in CASMCs than ASMCs were determined by the ß1 subunits. Moreover, Ca2+ sparks showed the similar effects on human CASMC and ASMC tone. In conclusions, Ca2+ sparks decrease CASMC tone and increase ASMC tone, mediated by BKs and Clca channels, respectively, and finally determined by LVDCCs.