Lipid droplet (LD) formation is a hallmark of cellular stress. Cells attempt to combat noxious stimuli by switching their metabolism from oxidative phosphorylation to glycolysis, sparing resources in LDs for generating cellular reducing power and for anabolic biosynthesis. Membrane phospholipids are also a source of LDs. To elucidate the formation of LDs, we exposed mice to hyperoxia, hypoxia, myocardial ischemia, and sepsis induced by cecal ligation and puncture (CLP). All the above-mentioned stressors enhanced the formation of LDs, as assessed by transmission electron microscopy, with severe mitochondrial swelling. Disruption of mitochondria by depleting mitochondrial DNA (ρ0 cells) significantly augmented the formation of LDs, causing transcriptional activation of fatty acid biosynthesis and metabolic reprogramming to glycolysis. Heme oxygenase (HO)-1 counteracts CLP-mediated septic shock in mouse models. In HO-1-deficient mice, LD formation was not observed upon CLP, but a concomitant decrease in “LD-decorating proteins” was observed, implying a link between LDs and cytoprotective activity. Collectively, LD biogenesis during stress can trigger adaptive LD formation, which is dependent on mitochondrial integrity and HO-1 activity; this may be a cellular survival strategy, apportioning energy-generating substrates to cellular defense.
Aims: Sepsis, a systemic inflammatory response to infection, represents the leading cause of death in critically ill patients. However, the pathogenesis of sepsis remains incompletely understood. Carbon monoxide (CO), when administered at low physiologic doses, can modulate cell proliferation, apoptosis, and inflammation in pre-clinical tissue injury models, though its mechanism of action in sepsis remains unclear. Results: CO (250 ppm) inhalation increased the survival of C57BL/6J mice injured by cecal ligation and puncture (CLP) through the induction of autophagy, the down-regulation of pro-inflammatory cytokines, and by decreasing the levels of bacteria in blood and vital organs, such as the lung and liver. Mice deficient in the autophagic protein, Beclin 1 (Becn1+/−) were more susceptible to CLP-induced sepsis, and unresponsive to CO therapy, relative to their corresponding wild-type (Becn1+/+) littermate mice. In contrast, mice deficient in autophagic protein microtubule-associated protein-1 light chain 3B (LC3B) (Map1lc3b−/−) and their corresponding wild-type (Map1lc3b+/+) mice showed no differences in survival or response to CO, during CLP-induced sepsis. CO enhanced bacterial phagocytosis in Becn1+/+ but not Becn1+/− mice in vivo and in corresponding cultured macrophages. CO also enhanced Beclin 1-dependent induction of macrophage protein signaling lymphocyte-activation molecule, a regulator of phagocytosis. Innovation: Our findings demonstrate a novel protective effect of CO in sepsis, dependent on autophagy protein Beclin 1, in a murine model of CLP-induced polymicrobial sepsis. Conclusion: CO increases the survival of mice injured by CLP through systemic enhancement of autophagy and phagocytosis. Taken together, we suggest that CO gas may represent a novel therapy for patients with sepsis. Antioxid. Redox Signal. 20, 432–442.
Fas-mediated apoptosis is a crucial cellular event. Fas, the Fas-associated death domain, and caspase 8 form the death-inducing signaling complex (DISC). Activated caspase 8 mediates the extrinsic pathways and cleaves cytosolic BID. Truncated BID (tBID) translocates to the mitochondria, facilitates the release of cytochrome c, and activates the intrinsic pathways. However, the mechanism causing these DISC components to aggregate and form the complex remains unclear. We found that Cav-1 regulated Fas signaling and mediated the communication between extrinsic and intrinsic pathways. Shortly after hyperoxia (4 h), the colocalization and interaction of Cav-1 and Fas increased, followed by Fas multimer and DISC formation. Deletion of Cav-1 (Cav-1−/−) disrupted DISC formation. Further, Cav-1 interacted with BID. Mutation of Cav-1 Y14 tyrosine to phenylalanine (Y14F) disrupted the hyperoxia-induced interaction between BID and Cav-1 and subsequently yielded a decreased level of tBID and resistance to hyperoxia-induced apoptosis. The reactive oxygen species (ROS) scavenger N-acetylcysteine decreased the Cav-1–Fas interaction. Deletion of glutathione peroxidase-2 using siRNA aggravated the BID–Cav-1 interaction and tBID formation. Taken together, these results indicate that Cav-1 regulates hyperoxia/ROS-induced apoptosis through interactions with Fas and BID, probably via Fas palmitoylation and Cav-1 Y14 phosphorylation, respectively.
Hyperoxia; Fas; Caveolin-1; Apoptosis; Free radicals
Tescalcin (TESC) is an EF-hand calcium binding protein that is differentially expressed in several tissues, however it is not reported that the expression and functional roles of TESC in colorectal cancer. Levels of messenger RNA (mRNA) and protein expression of TESC in colorectal cancer tissues were assessed using RT-PCR, real time PCR, immunohistochemistry, and clinicopathologic analyses. Quantitative analysis of TESC levels in serum specimens was performed using sandwich ELISA. Colorectal cancer cells transfected with TESC small interfering RNA and short hairpin RNA were examined in cell proliferation assays, phospho-MAPK array, and mouse xenograft models. Here we demonstrated that TESC is overexpressed in colorectal cancer (CRC), but was not expressed in normal mucosa and premalignant dysplastic lesions. Furthermore, serum TESC levels were elevated in patients with CRC. Knockdown of TESC inhibited the Akt-dependent NF-κB pathway and decreased cell survival in vitro. Depletion of TESC reduced tumor growth in a CRC xenograft model. Thus, TESC is a potential diagnostic marker and oncotarget in colorectal cancer.
Tescalcin; colorectal cancer; cell growth; tumor growth, NF-κB
Lung epithelial cell death is a prominent feature of hyperoxic lung injury, and has been considered a very important underlying mechanism of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Here we report on a novel mechanism involved in epithelial cytoprotection and homeostasis after oxidative stress. p62 (sequestosome 1; SQSTM1) is a ubiquitously expressed cellular protein. It interacts with ubiquitinated proteins and autophagic marker light chain 3b (LC3b), thus mediating the degradation of selective targets. In this study, we explored the role of p62 in mitochondria-mediated cell death after hyperoxia. Lung alveolar epithelial cells demonstrate abundant p62 expression, and p62 concentrations are up-regulated by oxidative stress at both the protein and mRNA levels. The p62/LC3b complex interacts with Fas and truncated BID (tBID) physically. These interactions abruptly diminish after hyperoxia. The deletion of p62 robustly increases tBID and cleaved caspase-3, implying an antiapoptotic effect. This antiapoptotic effect of p62 is further confirmed by measuring caspase activities, cleaved poly ADP ribose polymerase, and cell viability. The deletion of the p62 PBI domain or the ubiquitin-associated domain both lead to elevated tBID, cleaved caspase-3, and significantly more cell death after hyperoxia. Moreover, p62 traffics in an opposite direction with LC3b after hyperoxia, leading to the dissociation of the p62/Cav-1/LC3b/BID complex. Subsequently, the LC3b-mediated lysosomal degradation of tBID is eliminated. Taken together, our data suggest that the p62/LC3b complex regulates lung alveolar epithelial cell homeostasis and cytoprotection after hyperoxia.
p62/SQSTM1; hyperoxia; tBid; LC3b; apoptosis
Collagen triple helix repeat-containing 1 (CTHRC1) is known to be aberrantly upregulated in most human solid tumors, although the functional roles of CTHRC1 in colorectal cancer remain unclear. In this study, we investigated the occurrence of CTHRC1 upregulation and its role in vivo and in vitro. The expression profile and clinical importance of CTHRC1 were examined by reverse transcription-polymerase chain reaction and immunohistochemical analyses in normal and tumor patient samples. CTHRC1 was detectable in normal tissues, but also was highly expressed in tumor specimens. CTHRC1 upregulation was significantly associated with demethylation of the CTHRC1 promoter in colon cancer cell lines and tumor tissues. Clinicopathologic analyses showed that nodal status and expression of CTHRC1 (95% CI 0.999–3.984, p=0.05) were significant prognostic factors for disease-free survival. Promoter CpG methylation and hypermethylation status were measured by bisulfite sequencing and pyrosequencing analysis. Furthermore, we showed that overexpression of CTHRC1 in the SW480 and HT-29 cell lines increased invasiveness, an effect mediated by extracellular signal-regulated kinase (ERK)-dependent upregulation of matrix metalloproteinase 9 (MMP9). Consistent with this, we found that knockdown of CTHRC1 attenuated ERK activation and cancer cell invasivity. These results demonstrate that CTHRC1 expression is elevated in human colon cancer cell lines and clinical specimens, and promotes cancer cell invasivity through ERK-dependent induction of MMP9 expression. Our results further suggest that high levels of CTHRC1 expression are associated with poor clinical outcomes.
CTHRC1; ERK; MMP9; Invasion; Colorectal cancer
Thioredoxin-interacting protein (TXNIP) has multiple functions, including tumor suppression and involvement in cell proliferation and apoptosis. However, its role in the inflammatory process remains unclear. In this report, we demonstrate that Txnip−/− mice are significantly more susceptible to lipopolysaccharide (LPS)-induced endotoxic shock. In response to LPS, Txnip−/− macrophages produced significantly higher levels of nitric oxide (NO) and inducible nitric oxide synthase (iNOS), and an iNOS inhibitor rescued Txnip−/− mice from endotoxic shock-induced death, demonstrating that NO is a major factor in TXNIP-mediated endotoxic shock. This susceptibility phenotype of Txnip−/− mice occurred despite reduced IL-1β secretion due to increased S-nitrosylation of NLRP3 compared to wild-type controls. Taken together, these data demonstrate that TXNIP is a novel molecule that links NO synthesis and NLRP3 inflammasome activation during endotoxic shock.
TXNIP has many biological functions, including the inhibition of tumor growth, suppression of hepatocarcinogenesis, and regulation of glucose metabolism and reactive oxygen species (ROS) generation in different cell types. However, little is known about its role in the inflammatory process. In this study, our results demonstrate that TXNIP plays a critical role in the control of lethal endotoxin-induced shock by controlling NO production in innate immune cells via the regulation of iNOS expression. This regulation is mediated through changes in the activation and translocation of NF-κB that affect the NF-κB/iNOS pathway. In addition, excessive NO reduces the production of IL-1β via S-nitrosylation of the NLRP3 inflammasome. Subsequently, the survival of Txnip−/− mice is significantly decreased due to hypothermia and hypoglycemia. Overall, these results suggest that TXNIP is a novel therapeutic target for the treatment of inflammatory diseases.
Epithelial cell death plays a critical role in hyperoxia-induced lung injury. We investigated the involvement of the autophagic marker microtubule-associated protein-1 light chain-3B (LC3B) in epithelial cell apoptosis after hyperoxia. Prolonged hyperoxia (>95% O2), which causes characteristic lung injury in mice, activated morphological and biochemical markers of autophagy. Hyperoxia induced the time-dependent expression and conversion of LC3B-I to LC3B-II in mouse lung in vivo and in cultured epithelial cells (Beas-2B, human bronchial epithelial cells) in vitro. Hyperoxia increased autophagosome formation in Beas-2B cells, as evidenced by electron microscopy and increased GFP-LC3 puncta. The augmented LC3B level after hyperoxia was transcriptionally regulated and dependent in part on the c-Jun N-terminal kinase pathway. We hypothesized that LC3B plays a regulatory role in hyperoxia-induced epithelial apoptosis. LC3B siRNA promoted hyperoxia-induced cell death in epithelial cells, whereas overexpression of LC3B conferred cytoprotection after hyperoxia. The autophagic protein LC3B cross-regulated the Fas apoptotic pathway by physically interacting with the components of death-inducing signaling complex. This interaction was mediated by caveolin-1 tyrosine 14, which is a known target of phosphorylation induced by hyperoxia. Taken together, hyperoxia-induced LC3B activation regulates the Fas apoptotic pathway and thus confers cytoprotection in lung epithelial cells. The interaction of LC3B and Fas pathways requires cav-1.
apoptosis; autophagy; hyperoxia; lung injury; caveolin-1
Heme oxygenase (HO)-1 is a cytoprotective molecule that is induced during the response to injury. An increase in HO-1 is an acute indicator of inflammation, and early induction of HO-1 has been suggested to correlate with severity of injury. While a great deal is known about the induction of HO-1 by inflammatory mediators and bacterial lipopolysaccharide (LPS), much less is known about the effects of anti-inflammatory mediators on HO-1 expression. Transforming growth factor (TGF)-β is known to play a critical role in suppressing the immune response, and the TGF-β1 isoform is expressed in inflammatory cells. Thus, we wanted to investigate whether TGF-β1 could inhibit the expression of HO-1 during exposure to an inflammatory stimulus in macrophages. Here we demonstrate that TGF-β1 is able to downregulate LPS-induced HO-1 in mouse macrophages, and this reduction in HO-1 occurred through signaling of TGF-β1 via its type I receptor, and activation of Smad2. This TGF-β1 response is dependent on an intact Ets-binding site (EBS) located 93 base pairs upstream from the mouse HO-1 transcription start site. This EBS is known to be important for Ets-2 transactivation of HO-1 by LPS stimulation, and we show that TGF-β1 is able to suppress LPS-induced Ets-2 mRNA and protein levels in macrophages. Moreover, silencing of Smad2 is able to prevent the suppression of both HO-1 and Ets-2 by TGF-β1 during exposure to LPS. These data suggest that the return of HO-1 to basal levels during the resolution of an inflammatory response may involve its downregulation by anti-inflammatory mediators.
gene regulation; inflammatory response; cytoprotective molecule
Nucleotide-binding oligomerization domain protein 2 (NOD2) stimulates diverse inflammatory responses resulting in differential cellular phenotypes. To identify the role of NOD2 in vascular arterial obstructive diseases, we investigated the expression and pathophysiological role of NOD2 in a vascular injury model of neointimal hyperplasia.
Methods and Results
We first analyzed for neointimal hyperplasia following femoral artery injury in NOD2+/+ and NOD2−/− mice. NOD2−/− mice showed a 2.86-fold increase in neointimal formation that was mainly composed of SM α-actin positive cells. NOD2 was expressed in vascular smooth muscle cells (VSMCs) and NOD2−/− VSMCs showed increased cell proliferation in response to mitogenic stimuli, PDGF-BB or fetal bovine serum (FBS), compared with NOD2+/+ VSMCs. Furthermore, NOD2 deficiency markedly promoted VSMCs migration in response to PDGF-BB and this increased cell migration was attenuated by a PI3 kinase inhibitor. However, PKC and JNK inhibitors exerted negligible effects. Moreover, muramyl dipeptide-stimulated NOD2 prevented PDGF-BB-induced VSMCs migration.
Functional NOD2 is expressed in VSMCs, and NOD2 deficiency promoted VSMCs proliferation, migration, and neointimal formation after vascular injury. These results provide evidence for the involvement of NOD2 in vascular homeostasis and tissue injury, serving as a potential molecular target in the modulation of arteriosclerotic vascular disease.
Autophagy, an autodigestive process that degrades cellular organelles and protein, plays an important role in maintaining cellular homeostasis during environmental stress. Carbon monoxide (CO), a toxic gas and candidate therapeutic molecule, confers cytoprotection in animal models of acute lung injury. The mechanisms underlying CO-dependent lung cell protection and the role of autophagy in this process remain unclear. Here, we demonstrate that CO exposure time-dependently increased the expression and activation of the autophagic protein, microtubule-associated protein–1 light chain-3B (LC3B) in mouse lung, and in cultured human alveolar (A549) or human bronchial epithelial cells. Furthermore, CO increased autophagosome formation in epithelial cells by electron microscopy and green fluorescent protein (GFP)-LC3 puncta assays. Recent studies indicate that reactive oxygen species (ROS) play an important role in the activation of autophagy. CO up-regulated mitochondria-dependent generation of ROS in epithelial cells, as assayed by MitoSOX fluorescence. Furthermore, CO-dependent induction of LC3B expression was inhibited by N-acetyl-L-cysteine and the mitochondria-targeting antioxidant, Mito-TEMPO. These data suggest that CO promotes the autophagic process through mitochondrial ROS generation. We investigated the relationships between autophagic proteins and CO-dependent cytoprotection using a model of hyperoxic stress. CO protected against hyperoxia-induced cell death, and inhibited hyperoxia-associated ROS production. The ability of CO to protect against hyperoxia-induced cell death and caspase-3 activation was compromised in epithelial cells infected with LC3B-small interfering (si)RNA, indicating a role for autophagic proteins. These studies uncover a new mechanism for the protective action of CO, in support of potential therapeutic application of this gas.
apoptosis; autophagy; carbon monoxide; epithelial cells; hyperoxia
Rationale: Pulmonary hypertension (PH) is a progressive disease with unclear etiology. The significance of autophagy in PH remains unknown.
Objectives: To determine the mechanisms by which autophagic proteins regulate tissue responses during PH.
Methods: Lungs from patients with PH, lungs from mice exposed to chronic hypoxia, and human pulmonary vascular cells were examined for autophagy using electron microscopy and Western analysis. Mice deficient in microtubule-associated protein-1 light chain-3B (LC3B−/−), or early growth response-1 (Egr-1−/−), were evaluated for vascular morphology and hemodynamics.
Measurements and Main Results: Human PH lungs displayed elevated lipid-conjugated LC3B, and autophagosomes relative to normal lungs. These autophagic markers increased in hypoxic mice, and in human pulmonary vascular cells exposed to hypoxia. Egr-1, which regulates LC3B expression, was elevated in PH, and increased by hypoxia in vivo and in vitro. LC3B−/− or Egr-1−/−, but not Beclin 1+/−, mice displayed exaggerated PH during hypoxia. In vitro, LC3B knockdown increased reactive oxygen species production, hypoxia-inducible factor-1α stabilization, and hypoxic cell proliferation. LC3B and Egr-1 localized to caveolae, associated with caveolin-1, and trafficked to the cytosol during hypoxia.
Conclusions: The results demonstrate elevated LC3B in the lungs of humans with PH, and of mice with hypoxic PH. The increased susceptibility of LC3B−/− and Egr-1−/− mice to hypoxia-induced PH and increased hypoxic proliferation of LC3B knockdown cells suggest adaptive functions of these proteins during hypoxic vascular remodeling. The results suggest that autophagic protein LC3B exerts a protective function during the pathogenesis of PH, through the regulation of hypoxic cell proliferation.
autophagy; hypoxia; hypertension, pulmonary
Autophagy, a cellular process for organelle and protein turnover, regulates innate immune responses. We demonstrate that depletion of autophagic proteins microtubule associated protein-1 light chain 3B (LC3B) and Beclin 1 enhances caspase-1 activation and secretion of interleukin-1β and interleukin-18. Autophagic protein depletion promoted accumulation of dysfunctional mitochondria and cytosolic translocation of mitochondrial DNA (mtDNA) in response to lipopolysaccharide (LPS) and ATP in macrophages. Release of mtDNA into the cytosol depended on the NALP3 inflammasome and mitochondrial ROS. Cytosolic mtDNA contributed to IL-1β and IL-18 secretion in response to LPS and ATP. LC3B-deficient mice produced more caspase-1-dependent cytokines in two sepsis models and were susceptible to LPS-induced mortality. Our study suggests that autophagic proteins regulate NALP3-dependent inflammation by preserving mitochondrial integrity.
The molecular and cellular mechanisms underlying the pathogenesis of chronic obstructive pulmonary disease (COPD) remain incompletely understood. We have investigated the potential role of macro-autophagy, a cellular homeostatic mechanism, in COPD and cigarette smoke-induced lung-cell injury. Autophagy is a dynamic process for the turnover of organelles and proteins, which regenerates metabolic precursors through the lysosomal-dependent catabolism of cellular macromolecules. It is typically associated with survival pathways, especially in nutrient deficiency states. The role of autophagy in human diseases is less clear, and has been associated with both protective and detrimental consequences, depending on the disease model. While autophagy is considered cytoprotective, this process is often found in association with cell death, and the relationships between autophagy and cell death remain ambiguous. We have found elevated autophagy in COPD lung specimens, as well as in response to cigarette smoke exposure in vitro and in vivo. In our studies, the activation of autophagic proteins was associated with epithelial cell apoptosis in response to cigarette smoke, with pathogenic implications in COPD. Further studies are needed to determine the functional significance of autophagy in COPD and other diseases of the lung.
apoptosis; autophagy; chronic obstructive pulmonary disease; cigarette smoke; emphysema
Beclin 1, a tumor suppressor protein, acts as an initiator of autophagy in mammals. Heterozygous disruption of Beclin 1 accelerates tumor growth, but the underlying mechanisms remain unclear. We examined the role of Beclin 1 in tumor proliferation and angiogenesis, using a primary mouse melanoma tumor model. Beclin 1 (Becn1+/−) hemizygous mice displayed an aggressive tumor growth phenotype with increased angiogenesis under hypoxia, associated with enhanced levels of circulating erythropoietin but not vascular endothelial growth factor, relative to wild-type mice. Using in vivo and ex vivo assays, we demonstrated increased angiogenic activity in Becn1+/− mice relative to wild-type mice. Endothelial cells from Becn1+/− mice displayed increased proliferation, migration and tube formation in response to hypoxia relative to wild-type cells. Moreover, Becn1+/− cells subjected to hypoxia displayed increased hypoxia-inducible factor-2α (HIF-2α) expression relative to HIF-1α. Genetic interference of HIF-2α but not HIF-1α, dramatically reduced hypoxia-inducible proliferation, migration and tube formation in Becn1+/− endothelial cells. We demonstrated that mice deficient in the autophagic protein Beclin 1 display a pro-angiogenic phenotype associated with the upregulation of HIF-2α and increased erythropoietin production. These results suggest a relationship between Beclin 1 and the regulation of angiogenesis, with implications in tumor growth and development.
angiogenesis; autophagy; beclin 1; hypoxia-inducible factor
The extrinsic apoptotic pathway initiates when a death ligand, such as the Fas ligand, interacts with its cell surface receptor (ie., Fas/CD95), forming a death-inducing signaling complex (DISC). The Fas-dependent apoptotic pathway has been implicated in several models of lung or vascular injury. Carbon monoxide, an enzymatic product of heme oxygenase-1, exerts antiapoptotic effects at low concentration in vitro and in vivo.
Using mouse lung endothelial cells (MLEC), we examined the antiapoptotic potential of carbon monoxide against apoptosis induced by the Fas/CD95-activating antibody (Jo2). Carbon monoxide was applied to cell cultures in vitro. The expression and/or activation of apoptosis-related proteins and signaling intermediates were determined using Western Immunoblot and co-immunoprecipitation assays. Cell death was monitored by lactate dehydrogenase (LDH) release assays. Statistical significance was determined by student T-test and a value of P < 0.05 was considered significant.
Treatment of MLEC with Fas-activating antibody (Jo2) induced cell death associated with the formation of the DISC, and activation of caspases (-8, -9, and -3), as well as the pro-apoptotic Bcl-2 family protein Bax. Exposure of MLEC to carbon monoxide inhibited Jo2-induced cell death, which correlated with the inhibition of DISC formation, cleavage of caspases-8, -9, and -3, and Bax activation. Carbon monoxide inhibited the phosphorylation of the Fas-associated death domain-containing protein, as well as its association with the DISC. Furthermore, carbon monoxide induced the expression of the antiapoptotic protein FLIP and increased its association with the DISC.
CO-dependent cytoprotection against Fas mediated apoptosis in MLEC depended in part on activation of ERK1/2-dependent signaling.
Carbon monoxide has been proposed as a potential therapy for lung and other diseases based in part on its antiapoptotic effects in endothelial cells. In vitro, carbon monoxide may inhibit both Fas/caspase-8 and Bax-dependent apoptotic signaling pathways induced by Fas-activating antibody in endothelial cells. Strategies to block Fas-dependent apoptotic pathways may be useful in development of therapies for lung or vascular disorders.
Autophagy, or “self eating,” refers to a regulated cellular process for the lysosomal-dependent turnover of organelles and proteins. During starvation or nutrient deficiency, autophagy promotes survival through the replenishment of metabolic precursors derived from the degradation of endogenous cellular components. Autophagy represents a general homeostatic and inducible adaptive response to environmental stress, including endoplasmic reticulum stress, hypoxia, oxidative stress, and exposure to pharmaceuticals and xenobiotics. Whereas elevated autophagy can be observed in dying cells, the functional relationships between autophagy and programmed cell death pathways remain incompletely understood. Preclinical studies have identified autophagy as a process that can be activated during vascular disorders, including ischemia–reperfusion injury of the heart and other organs, cardiomyopathy, myocardial injury, and atherosclerosis. The functional significance of autophagy in human cardiovascular disease pathogenesis remains incompletely understood, and potentially involves both adaptive and maladaptive outcomes, depending on model system. Although relatively few studies have been performed in the lung, our recent studies also implicate a role for autophagy in chronic lung disease. Manipulation of the signaling pathways that regulate autophagy could potentially provide a novel therapeutic strategy in the prevention or treatment of human disease.
autophagy; apoptosis; vascular disease
Chronic obstructive pulmonary disease (COPD) is a progressive lung disease characterized by abnormal cellular responses to cigarette smoke, resulting in tissue destruction and airflow limitation. Autophagy is a degradative process involving lysosomal turnover of cellular components, though its role in human diseases remains unclear.
Methodology and Principal Findings
Increased autophagy was observed in lung tissue from COPD patients, as indicated by electron microscopic analysis, as well as by increased activation of autophagic proteins (microtubule-associated protein-1 light chain-3B, LC3B, Atg4, Atg5/12, Atg7). Cigarette smoke extract (CSE) is an established model for studying the effects of cigarette smoke exposure in vitro. In human pulmonary epithelial cells, exposure to CSE or histone deacetylase (HDAC) inhibitor rapidly induced autophagy. CSE decreased HDAC activity, resulting in increased binding of early growth response-1 (Egr-1) and E2F factors to the autophagy gene LC3B promoter, and increased LC3B expression. Knockdown of E2F-4 or Egr-1 inhibited CSE-induced LC3B expression. Knockdown of Egr-1 also inhibited the expression of Atg4B, a critical factor for LC3B conversion. Inhibition of autophagy by LC3B-knockdown protected epithelial cells from CSE-induced apoptosis. Egr-1−/− mice, which displayed basal airspace enlargement, resisted cigarette-smoke induced autophagy, apoptosis, and emphysema.
We demonstrate a critical role for Egr-1 in promoting autophagy and apoptosis in response to cigarette smoke exposure in vitro and in vivo. The induction of autophagy at early stages of COPD progression suggests novel therapeutic targets for the treatment of cigarette smoke induced lung injury.