Cigarette smoke induces Akt protein degradation by the ubiquitin-proteasome system

Emphysema is one of the characteristic features of chronic obstructive pulmonary disease, which is caused mainly by cigarette smoking. Recent data have suggested that apoptosis and cell cycle arrest may contribute to the development of emphysema. In this study, we addressed the question of whether and how cigarette smoke affected Akt, which plays a critical role in cell survival and proliferation. In normal human lung fibroblasts, cigarette smoke extract (CSE) caused cell death, accompanying degradation of total and phosphorylated Akt (p-Akt), which was inhibited by MG132. CSE exposure resulted in preferential ubiquitination of the active Akt (myristoylated), rather than the inactive (T308A/S473A double mutant) Akt. Consistent with cytotoxicity, CSE induced a progressive decrease of phosphorylated human homolog of mouse double minute homolog 2 (p-HDM2) and phosphorylated apoptosis signal regulating kinase 1 (p-ASK1) with concomitant elevation of p53, p21, and phosphorylated p38 MAPK. Forced expression of the active Akt reduced both CSE-induced cytotoxicity and alteration in HDM2/p53/p21 and ASK1/p38 MAPK, compared with the inactive Akt. Of note, CSE induced expression of the tetratrico-peptide repeat domain 3 (TTC3), known as a ubiquitin ligase for active Akt. TTC3 siRNAs suppressed not only CSE-induced Akt degradation but also CSE-induced cytotoxicity. Accordingly, rat lungs exposed to cigarette smoke for 3 months showed elevated TTC3 expression and reduced Akt and p-Akt. Taken together, these data suggest that cigarette smoke induces cytotoxicity, partly through Akt degradation via the ubiquitin-proteasome system, in which TTC3 acts as a ubiquitin ligase for active Akt.     

Bone marrow cells repair cigarette smoke-induced emphysema in rats

The therapeutic potential of stem cells in chronic obstructive pulmonary disease is not well known although stem cell therapy is effective in models of other pulmonary diseases. We tested the capacities of bone marrow cells (BMCs), mesenchymal stem cells (MSCs), and conditioned media of MSCs (MSC-CM) to repair cigarette smoke-induced emphysema. Inbred female Lewis rats were exposed to cigarette smoke for 6 mo and then received BMCs, MSCs, or MSC-CM from male Lewis rats. For 2 mo after injection, the BMC treatment gradually alleviated the cigarette smoke-induced emphysema and restored the increased mean linear intercept. The BMC treatment significantly increased cell proliferation and the number of small pulmonary vessels, reduced apoptotic cell death, attenuated the mean pulmonary arterial pressure, and inhibited muscularization in small pulmonary vessels. However, only a few male donor cells were detected from 1 day to 1 mo after BMC administration. The MSCs and cell-free MSC-CM also induced the repair of emphysema and increased the number of small pulmonary vessels. Our data show that BMC, MSCs, and MSC-CM treatment repaired cigarette smoke-induced emphysema. The repair activity of these treatments is consistent with a paracrine effect rather than stem cell engraftment because most of the donor cells disappeared and because cell-free MSC-CM also induced the repair.     

Protein kinase C alpha and zeta differentially regulate death-inducing signaling complex formation in cigarette smoke extract-induced apoptosis

Cigarette smoke, a major risk factor in emphysema, causes cell death by incompletely understood mechanisms. Death-inducing signaling complex (DISC) formation is an initial event in Fas-mediated apoptosis. We demonstrate that cigarette smoke extract (CSE) induces DISC formation in human lung fibroblasts (MRC-5) and promotes DISC trafficking from the Golgi complex to membrane lipid rafts. We demonstrate a novel role of protein kinase C (PKC) in the regulation of DISC formation and trafficking. The PKC isoforms, PKCalpha, zeta, epsilon, and eta, were activated by CSE exposure. Overexpression of wild-type PKCalpha inhibited, while PKCzeta promoted, CSE-induced cell death. Dominant-negative (dn)PKCzeta protected against CSE-induced cell death by suppressing DISC formation and caspase-3 activation, while dnPKCalpha enhanced cell death by promoting these events. DISC formation was augmented by wortmannin, an inhibitor of PI3K. CSE-induced Akt phosphorylation was reduced by dnPKCalpha, but it was increased by dnPKCzeta. Expression of PKCalpha in vivo inhibited DISC formation, caspase-3/8 activation, lung injury, and cell death after prolonged cigarette smoke exposure, whereas expression of PKCzeta promoted caspase-3 activation. In conclusion, CSE-induced DISC formation is differentially regulated by PKCalpha and PKCzeta via the PI3K/Akt pathway. These results suggest that modulation of PKC may have therapeutic potential in the prevention of smoke-related lung injury.     

Cigarette smoke extract inhibits angiogenesis of pulmonary artery endothelial cells: the role of calpain

Angiogenesis is an integral part of both the pulmonary inflammatory response to chronic exposure to cigarette smoke and the lung tissue remodeling associated with cigarette smoke-induced chronic obstructive pulmonary disease (COPD). To investigate the role of angiogenesis in the pathogenesis of COPD, we evaluated the effect of cigarette smoke extract (CSE) on angiogenesis of pulmonary artery endothelial cells (PAEC). Incubation of PAEC with 2.5-10% CSE resulted in a dose-dependent inhibition of endothelial monolayer wound repair. CSE also caused inhibition of tube formation on Matrigel, migration in a Boyden chamber, and proliferation of PAEC. Because calpain, a family of calcium-dependent intracellular proteases, mediates cytoskeletal signaling in endothelial motility, we explored the role of calpain in the CSE-induced inhibition of endothelial angiogenesis. Incubation of CSE resulted in a dose-dependent decrease in calpain activity. Calpain inhibitor-1, a specific inhibitor of calpain, potentiates inhibitory effect of CSE on the endothelial monolayer wound repair, tube formation, cell migration, and cell proliferation. Transfection of PAEC with antisense oligodeoxyribonucleotides of calpastatin, the major endogenous calpain inhibitor, prevented CSE-induced increase in calpastatin protein content and CSE-induced decreases in calpain activity. It also prevented CSE-induced decreases in monolayer wound repair, tube formation, and migration. These results suggest that CSE attenuates angiogenesis of PAEC and the mechanism involves inhibition of calpain. Impaired angiogenesis may impede the repair process in the lungs of cigarette smokers and contribute to the altered structural remodeling observed in the lungs of patients with cigarette smoke-related COPD.     

STAT3 activation inhibits human bronchial epithelial cell apoptosis in response to cigarette smoke exposure

We have previously reported that cigarette smoke can induce DNA damage in human lung cells without leading to apoptosis or necrosis. In this study, we report that STAT3 is required for the survival of human bronchial epithelial cells (HBECs) following cigarette smoke-induced DNA damage. Cigarette smoke extract (CSE) exposure increases STAT3 phosphorylation (Tyr 705) and DNA binding activity in HBECs. CSE also stimulates IL-6 release and mRNA expression. Anti-IL-6 neutralizing antibody partially blocks STAT3 activation and renders the cells sensitive to CSE-induced DNA damage. Suppression of STAT3 by siRNA results in severe DNA damage and cell death in response to CSE exposure. These findings suggest that STAT3 mediates HBEC survival in response to CSE-induced DNA damage, at least in part, through the IL-6/STAT3 signaling pathway.     

Vitamin C prevents cigarette smoke-induced oxidative damage in vivo

Our recent in vitro results [4] indicate that cigarette smoke induces oxidation of human plasma proteins and extensive oxidative degradation of the guinea pig lung, heart, and liver microsomal proteins, which is almost completely prevented by ascorbic acid. In this paper, we substantiate the in vitro results with in vivo observations. We demonstrate that exposure of subclinical or marginal vitamin C-deficient guinea pigs to cigarette smoke causes oxidation of plasma proteins as well as extensive oxidative degradation of the lung microsomal proteins. Cigarette smoke exposure also results in some discernible damage of the heart microsomal proteins. The oxidative damage has been manifested by SDS-PAGE, accumulation of carbonyl and bityrosine, as well as loss of tryptophan and protein thiols. Cigarette smoke exposure also induces peroxidation of microsomal lipids as evidenced by the formation of conjugated dienes, malondialdehyde, and fluorescent pigment. Cigarette smoke-induced oxidative damage of proteins and peroxidation of lipids are accompanied by marked drop in the tissue ascorbate levels. Protein damage and lipid peroxidation are also observed in cigarette smoke-exposed pair-fed guinea pigs receiving 5 mg vitamin C/animal/day. However, complete protection against protein damage and lipid peroxidation occurs when the guinea pigs are fed 15 mg vitamin C/animal/day. Also, the cigarette smoke-induced oxidative damage of proteins and lipid is reversed after discontinuation of cigarette smoke exposure accompanied by ascorbate therapy. The results, if extrapolated to humans, indicate that comparatively large doses of vitamin C may protect the smokers from cigarette smoke-induced oxidative damage and associated degenerative diseases.     

MAPK pathway mediates EGR-1-HSP70-dependent cigarette smoke-induced chemokine production

Cigarette smoking, a major risk factor for chronic obstructive pulmonary disease, can cause airway inflammation, airway narrowing, and loss of elasticity, leading to chronic airflow limitation. In this report, we sought to define the signaling pathways activated by smoke and to identify molecules responsible for cigarette smoke-induced inflammation. We applied cigarette smoke water extract (CSE) to primary human lung fibroblasts and found that CSE significantly increased CXC chemokine IL-8 production. Meanwhile, 70-kDa heat shock protein (HSP70) was also induced by CSE in a dose- and time-dependent manner. CSE treatment stimulated HSP70 secretion by primary fibroblasts, which augmented IL-8 production. This was further confirmed by exogenously added recombinant HSP70. Using HSP70 small interfering RNA, we confirmed that CSE-induced chemokine production was dependent on heat shock protein expression. Further investigation showed that CSE could also stimulate early growth response-1 (EGR-1) in an ERK-dependent manner and that the expression of HSP70 was EGR-1 dependent. In view of these findings, we hypothesize that the MAPK-EGR-1-HSP70 pathway regulates the cigarette smoke-induced inflammatory process.     

Anti-malarial drug artesunate protects against cigarette smoke-induced lung injury in mice

Cigarette smoking is the primary cause of chronic obstructive pulmonary disease (COPD), which is mediated by lung infiltration with inflammatory cells, enhanced oxidative stress, and tissue destruction. Anti-malarial drug artesunate has been shown to possess anti-inflammatory and anti-oxidative actions in mouse asthma models. We hypothesized that artesunate can protect against cigarette smoke-induced acute lung injury via its anti-inflammatory and anti-oxidative properties. Artesunate was given by oral gavage to BALB/c mice daily 2 h before 4% cigarette smoke exposure for 1 h over five consecutive days. Bronchoalveolar lavage (BAL) fluid and lungs were collected for analyses of cytokines, oxidative damage and antioxidant activities. Bronchial epithelial cell BEAS-2B was exposed to cigarette smoke extract (CSE) and used to study the mechanisms of action of artesunate. Artesunate suppressed cigarette smoke-induced increases in BAL fluid total and differential cell counts; levels of IL-1β, MCP-1, IP-10 and KC; and levels of oxidative biomarkers 8-isoprostane, 8-OHdG and 3-nitrotyrosine in a dose-dependent manner. Artesunate promoted anti-oxidant catalase activity and reduced NADPH oxidase 2 (NOX2) protein level in the lungs from cigarette smoke-exposed mice. In BEAS-2B cells, artesunate suppressed pro-inflammatory PI3 K/Akt and p44/42 MAPK signaling pathways, and increased nuclear Nrf2 accumulation in response to CSE. Artesunate possesses anti-inflammatory and anti-oxidative properties against cigarette smoke-induced lung injury, probably via inhibition of PI3K and p42/22 MAPK signaling pathways, augmentation of Nrf2 and catalase activities, and reduction of NOX2 level. Our data suggest that artesunate may have therapeutic potential for treating COPD.     

Exposure to Biomass Smoke Extract Enhances Fibronectin Release from Fibroblasts

COPD induced following biomass smoke exposure has been reported to be associated with a more fibrotic phenotype than cigarette smoke induced COPD. This study aimed to investigate if biomass smoke induced extracellular matrix (ECM) protein production from primary human lung fibroblasts in vitro. Primary human lung fibroblasts (n = 5–10) were stimulated in vitro for up to 72 hours with increasing concentrations of biomass smoke extract (BME) or cigarette smoke extract (CSE) prior to being assessed for deposition of ECM proteins, cytokine release, and activation of intracellular signalling molecules. Deposition of the ECM proteins perlecan and fibronectin was upregulated by both CSE (p<0.05) and BME (p<0.05). The release of the neutrophilic chemokine IL-8 was also enhanced by BME. ERK1/2 phosphorylation was significantly upregulated by BME (p<0.05). Chemical inhibition of ERK signalling molecules partially attenuated these effects (p<0.05). Stimulation with endotoxin had no effect. This study demonstrated that BME had similar effects to CSE in vitro and had the capacity to directly induce fibrosis by upregulating production of ECM proteins. The mechanisms by which both biomass and cigarette smoke exposure cause lung damage may be similar.     

LncRNA LCPAT1 is involved in DNA damage induced by CSE

Cigarette smoking plays an important role in the process of lung cancer, during which DNA damage is proved to be involved. Non-coding RNAs are found to be involved in the DNA damage and repair processes induced by cigarette smoke. In the present study, we investigated the role of lncRNA LCPAT1 in DNA damage caused by CSE in Beas-2B cells. Our results indicate that LCPAT1, through RCC2 is involved in the CSE-induced DNA damage providing new insight into the lung carcinogenesis related to cigarette smoking.     

Cigarette smoke-induced alveolar epithelial–mesenchymal transition is mediated by Rac1 activation

Background

Epithelial–mesenchymal transition (EMT) is the major pathophysiological process in lung fibrosis observed in chronic obstructive pulmonary disease (COPD) and lung cancer. Smoking is a risk factor for developing EMT, yet the mechanism remains largely unknown. In this study, we investigated the role of Rac1 in cigarette smoke (CS) induced EMT.

Methods

EMT was induced in mice and pulmonary epithelial cells by exposure of CS and cigarette smoke extract (CSE) respectively.

Results

Treatment of pulmonary epithelial cells with CSE elevated Rac1 expression associated with increased TGF-β1 release. Blocking TGF-β pathway restrained CSE-induced changes in EMT-related markers. Pharmacological inhibition or knockdown of Rac1 decreased the CSE exposure induced TGF-β1 release and ameliorated CSE-induced EMT. In CS-exposed mice, pharmacological inhibition of Rac1 reduced TGF-β1 release and prevented aberrations in expression of EMT markers, suggesting that Rac1 is a critical signaling molecule for induction of CS-stimulated EMT. Furthermore, Rac1 inhibition or knockdown abrogated CSE-induced Smad2 and Akt (PKB, protein kinase B) activation in pulmonary epithelial cells. Inhibition of Smad2, PI3K (phosphatidylinositol 3-kinase) or Akt suppressed CSE-induced changes in epithelial and mesenchymal marker expression.

Conclusions and general significance

Altogether, these data suggest that CS initiates EMT through Rac1/Smad2 and Rac1/PI3K/Akt signaling pathway. Our data provide new insights into the fundamental basis of EMT and suggest a possible new course of therapy for COPD and lung cancer.     

Inhibition of PI3K prevents the proliferation and differentiation of human lung fibroblasts into myofibroblasts: the role of class I P110 isoforms

Idiopathic pulmonary fibrosis (IPF) is a progressive fibroproliferative disease characterized by an accumulation of fibroblasts and myofibroblasts in the alveolar wall. Even though the pathogenesis of this fatal disorder remains unclear, transforming growth factor-β (TGF-β)-induced differentiation and proliferation of myofibroblasts is recognized as a primary event. The molecular pathways involved in TGF-β signalling are generally Smad-dependent yet Smad-independent pathways, including phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt), have been recently proposed. In this research we established ex-vivo cultures of human lung fibroblasts and we investigated the role of the PI3K/Akt pathway in two critical stages of the fibrotic process induced by TGF-β: fibroblast proliferation and differentiation into myofibroblasts. Here we show that the pan-inhibitor of PI3Ks LY294002 is able to abrogate the TGF-β-induced increase in cell proliferation, in α- smooth muscle actin expression and in collagen production besides inhibiting Akt phosphorylation, thus demonstrating the centrality of the PI3K/Akt pathway in lung fibroblast proliferation and differentiation. Moreover, for the first time we show that PI3K p110δ and p110γ are functionally expressed in human lung fibroblasts, in addition to the ubiquitously expressed p110α and β. Finally, results obtained with both selective inhibitors and gene knocking-down experiments demonstrate a major role of p110γ and p110α in both TGF-β-induced fibroblast proliferation and differentiation. This finding suggests that specific PI3K isoforms can be pharmacological targets in IPF.     

Endothelin-1 promotes myofibroblast induction through the ETA receptor via a rac/phosphoinositide 3-kinase/Akt-dependent pathway and is essential for the enhanced contractile phenotype of fibrotic fibroblasts

The endothelins are a family of endothelium-derived peptides that possess a variety of functions, including vasoconstriction. Endothelin-1 (ET-1) is up-regulated during tissue repair and promotes myofibroblast contraction and migration, hence contributing to matrix remodeling during tissue repair. Here, we show that addition of ET-1 to normal lung fibroblasts induces expression of proteins that contribute to a contractile phenotype, including alpha-smooth muscle actin (alpha-SMA), ezrin, moesin, and paxillin. We confirm that ET-1 enhances the ability of lung fibroblasts to contract extracellular matrix, a function essential for tissue repair, through induction of de novo protein synthesis. Blockade of the Akt/phosphoinositide 3-kinase (PI3-kinase) pathway with LY294002 and wortmannin prevents the ability of ET-1 to induce alpha-SMA, ezrin, paxillin, and moesin and to promote matrix contraction. Dominant negative rac and Akt blocked the ability of ET-1 to promote formation of alpha-SMA stress fibers. Using specific ET-1 receptor inhibitors, we show that ET-1 induces collagen matrix contraction through the ETA, but not the ETB, receptor. Relative to normal pulmonary fibroblasts, fibroblasts cultured from scars of patients with the fibrotic disease systemic sclerosis (scleroderma) show enhanced ET-1 expression and binding. Systemic sclerosis lung fibroblasts show increased ability to contract a collagen matrix and elevated expression of the procontractile proteins alpha-SMA, ezrin, paxillin, and moesin, which are greatly reduced by antagonizing endogenous ET-1 signaling. Thus, blocking ET-1 or the PI3-kinase/Akt cascades might be beneficial in reducing scar formation in pulmonary fibrosis.     

Prostaglandin E(2) protects human lung fibroblasts from cigarette smoke extract-induced apoptosis via EP(2) receptor activation

Prostaglandin E(2) (PGE(2)) has been shown to have a strong cytoprotective effect, inhibiting apoptosis. In the present study, we evaluated whether PGE(2) has a protective effect on cigarette smoke extract (CSE)-induced apoptosis in human lung fibroblasts. Apoptosis was assessed by various methods, including DNA content analysis. CSE (15%-20%) led to apoptosis and induced imbalance in favor of pro- over anti-apoptotic protein expression and activated caspases. PGE(2) blocked CSE-induced apoptosis and modulated the balance of pro- and anti-apoptotic proteins and decreased the activation of caspases. This anti-apoptotic effect was mediated via EP(2) receptor activation as the EP(2) agonist butaprost mimicked PGE(2) activity and siRNA for the EP(2) receptor blocked it. An adenylyl cyclase inhibitor was found to abolish the PGE(2)-mediated cytoprotective effect. Correspondingly, c-AMP analogs blocked CSE-induced apoptosis. Consistently, the protein kinase A (PKA) inhibitor KT-5720 abolished PGE(2)-mediated protection. PGE(2) and butaprost phosphorylated Bad and KT-5720 blocked phosphorylation. These results suggest that PGE(2) inhibits CSE-induced apoptosis via EP(2) receptor activation and activation of PKA, which leads to an alteration in the balance between pro- and anti-apoptotic factors. Through such a mechanism, PGE(2) may alter survival of cells in the smoke-exposed lungs, thus affecting the pathogenesis of cigarette smoke-induced disease.     

Differential induction of apoptosis by cigarette smoke extract in primary human lung fibroblast strains: implications for emphysema

Cigarette smoke is the principal cause of emphysema. Recent attention has focused on the loss of alveolar fibroblasts in the development of emphysema. Fibroblasts may become damaged by oxidative stress and undergo apoptosis as a result of cigarette smoke exposure. Not all smokers develop lung diseases associated with tobacco smoke, a fact that may reflect individual variation among human fibroblast strains. We hypothesize that fibroblasts from different human beings vary in their ability to undergo apoptosis after cigarette smoke exposure. This could account for emphysematous changes that occur in the lungs of some but not all smokers. Primary human lung fibroblast strains were exposed to cigarette smoke extract (CSE) and assessed for viability, morphological changes, and mitochondrial transmembrane potential as indicators of apoptosis. We also examined the generation of intracellular reactive oxygen species (ROS), 4-hydroxy-2-nonenal, and changes in glutathione (GSH) and glutathione disulfide (GSSG) levels. Each human lung fibroblast strain exhibited a differential sensitivity to CSE as judged by changes in mitochondrial membrane potential, viability, ROS generation, and glutathione production. Interestingly, the thiol antioxidants N-acetyl-L-cysteine and GSH eliminated CSE-induced changes in fibroblast morphology such as membrane blebbing, nuclear condensation, and cell size and prevented alterations in mitochondrial membrane potential and the generation of ROS. These findings support the concept that oxidative stress and apoptosis are responsible for fibroblast death associated with exposure to tobacco smoke. Variations in the sensitivity of fibroblasts to cigarette smoke may account for the fact that only some smokers develop emphysema.     

Tobacco Smoke Activates Human Papillomavirus 16 p97 Promoter and Cooperates with High-Risk E6/E7 for Oxidative DNA Damage in Lung Cells

We have previously shown a functional interaction between human papillomavirus type 16 (HPV-16) E6 and E7 oncoproteins and cigarette smoke condensate (CSC) in lung cells suggesting cooperation during carcinogenesis. The molecular mechanisms of such interaction, however, remain to be elucidated. Here we first present evidence showing that cigarette smoke condensate (CSC) has the ability to activate the HPV-16 p97 promoter by acting on the long control region (LCR) in lung epithelial cells. Interestingly, we observed that CSC-induced p97 promoter activation occurs in a dose-dependent manner in both tumor A-549 (lung adenocarcinoma), H-2170 (bronchial carcinoma), SiHa or Hela (cervical carcinoma) cells but not in non-tumor BEAS-2B (bronchial) or NL-20 (alveolar) lung cells unless they ectopically expressed the HPV-16 E6 and E7 oncogenes. In addition, we also observed a significant increase of primary DNA damage in tumor and non-tumor CSC-treated lung cells expressing HPV-16 E6 and E7 oncogenes suggesting a cooperative effect in this process, even though the contribution of E7 was significantly higher. Taken together, our results strongly suggest that tobacco smoke is able to induce the activation of the HPV-16 p97 promoter in cooperation with HPV-16 E6 and E7 oncogenes that, in turn, sensitize lung cells to tobacco smoke-induced DNA damage.     

Regional heterogeneity in murine lung fibroblasts from normal mice or mice exposed once to cigarette smoke

Chronic obstructive lung disease (COPD) is characterized by matrix deposition in the small airways but matrix loss from the parenchyma, phenomena which must depend on the ability of local fibroblasts to produce matrix after smoke exposure. To investigate this idea, we exposed C57Bl/6 mice once to cigarette smoke or to air (control) and prepared primary cultures of lung fibroblasts by microdissecting large airways (trachea, LAF), medium size airways (major bronchi, MAF) and parenchyma (PF). Control PF showed the lowest rate of wound closure and wound closure was depressed in all lines by a single in vivo smoke exposure. Gene expression of matrix proteins differed considerably among the sites; decorin, which may sequester TGFβ, was markedly higher in PF. PF showed higher intrinsic ratios of pSmad2/Smad2. Smoke caused much greater increases in secreted and matrix deposited collagens 1 and 3 in PF than in LAF or MAF. Expression of Thy-1, a gene that suppresses myofibroblast differentiation, was increased by smoke in PF. We conclude that there is considerable regional heterogeneity in murine lung fibroblasts in terms of matrix production, either basally or after in vivo smoke exposure; that PF have lower ability to repair wounds and higher intrinsic TGFβ signaling; and that a single exposure to smoke produces lasting changes in the pattern of matrix production and wound repair, changes that may be mediated in part by smoke-induced release of TGFβ. However, PF still retain the ability to repair by producing new matrix after a single in vivo smoke exposure.