Effect of Phosphodiestrase 4 inhibitor against Cigarette Smoke Extract Induced Autophagy and Apoptosis in Human Lung
Fibroblasts (MRC-5 Cells)
가천대학교 의학전문대학원 내과학교실
박 정 웅
Purpose: Cigarette smoke (CS), a major causative agent of chronic obstructive pulmonary disease (COPD), induces lung
cell death by incompletely understood mechanisms. The induction of apoptosis in lung structural cells by CS may contribute to the pathogenesis of emphysema. Phosphodiesterase-4 (PDE4) inhibitors are anti-inflammatory agents used in COPD therapy that can prevent CS-induced emphysema in mice. We investigated the effect of Rolipram, a first generation PDE4 inhibitor, on the regulation of CS-induced apoptosis.
Methods: Human lung fibroblast (MRC-5) cells were exposed to cigarette smoke extract (CSE). Cell viability and apoptosis
were determined by MTT assay and Annexin-V staining, respectively. Nuclear morphology was determined by Hoechst staining. Caspase activation was determined by Western analysis.
Results: Rolipram protected against cell death and increased viability in MRC-5 fibroblasts after CSE exposure. Rolipram
protected against apoptosis, decreased caspase-3 and -8 cleavage, and prevented nuclear condensation and fragmentation in MRC-5 cells exposed to CSE. Pretreatment with Rolipram enhanced Akt phosphorylation and associated cytoprotecion in CSE-treated cells, which could be reversed by the PI3K inhibitor LY294002.
Conclusions: Rolipram protects against apoptosis of MRC-5 cells through inhibition of caspases-3 and -8. Rolipram may
represent an effective therapeutic agent to reduce CS-induced apoptosis of lung fibroblasts.
Key Words: Apoptosis; Cigarette Smoke; Chronic Obstructive Pulmonary Disease, PDE4 inhibitor.
INTRODUCTION
Chronic obstructive pulmonary disease (COPD) contrib- utes significantly to the global burden of disease. The etiology of COPD remains unclear, but involves aberrant inflammatory responses of the lung caused by chronic cigarette smoke (CS) or particle exposure, which can be aggravated by infections [1]. The complex pathology of this disease includes bronchitis associated with airway inflammation and mucous obstruction, as well as emphysema resulting from the loss of alveolar surface area for gas exchange. The mechanisms underlying alveolar destruction remain unclear, but include protease/ an- ti-protease imbalance, oxidative stress, and apoptosis [1-4].
The clinical management of COPD involves both pharma-
cological and non-pharmacological treatments. Long-acting bronchodilators such as long-acting β2 agonists and long-act- ing anti-muscarinic drugs are employed in the symptomatic management of COPD, yet have limited clinical efficacy [5].
Inhaled corticosteroids represent the main anti-inflammatory treatment used in COPD therapy despite a relatively modest clinical efficacy in this disease relative to asthma. Unlike asth- ma, inhaled corticosteroids are recommended only in the more advanced stages of COPD (i.e., stage III and IV, accord- ing to the Global Initiative for Chronic Obstructive Lung Disease [GOLD]), and are usually combined with long-acting β2 agonists in order to reduce the number of exacerbations [6,7].
Recently, phosphodiesterase-4 (PDE4) inhibitors have
been shown to provide therapeutic benefit, particularly in pa- tients with late stage COPD. Phosphodiesterases (PDEs) are cAMP- or cGMP-specific enzymes which have a ubiquitous distribution in most human cells. Eleven PDE isoenzymes have been identified to date [8]. Of these, PDE4, which hydrolyzes cAMP, is expressed in lung structural cells such as smooth muscle cells, airway epithelium, and inflammatory cells (i.e., neutrophils, lymphocytes, and macrophages) [9].
Phase III clinical studies have shown that the PDE4 inhibitor Roflumilast significantly improved post-bronchodilator FEV1 and reduced exacerbation rate, dyspnea severity, and the use of rescue medication, without affecting systemic inflammation, time-to-mortality, and health utility scores in the clinical phenotype of chronic bronchitis [10,11]. PDE4 inhibitors are known to mitigate mucociliary malfunction, lung fibrotic and emphysematous remodeling, and oxidative stress [12]. In vivo studies have confirmed that the PDE4 inhibitor Roflumilast can alleviate emphysema in mice exposed to chronic CS, and also reduce sub-epithelial collagen deposition in the airways of mice repetitively challenged with ovalbumin [12].
Apoptosis may represent an important factor in the patho- genesis of COPD, which contributes to emphysematous lung destruction in response to CS [3]. Lung tissues from patients with COPD displayed greater numbers of apoptotic cells than control lungs or lungs from smokers without COPD [3,13].
These findings suggest that inhibition of CS-induced apoptosis may have a beneficial effect in COPD progression.
However, the effects of PDE4 inhibitors on apoptosis of lung structural cells remain poorly understood in the context of COPD therapy. The PDE4 inhibitor Roflumilast can induce cAMP-dependent signaling pathways which have been shown to confer protection against cardiomyocyte apoptosis [14].
In the CS-induced emphysema model, the PDE4 inhibitor GPD-1116 attenuated emphysema by inhibiting CS-induced matrix metalloproteinase (MMP)-12 activity and protecting lung cells from apoptosis, but did not prevent the decrease of vascular endothelial growth factor [15]. We have previously shown that the xanthine derivatives (i.e., aminophylline or theophylline) protected against apoptosis of MRC-5 cells
through the inactivation of caspases -3 and -8 [16]. Therefore, the current study was designed to determine whether Rolipram, a PDE4 inhibitor, could protect against apoptosis of lung fibroblasts in response to exposure to aqueous cigarette smoke extract (CSE), by directly inhibiting the extrinsic apop- totic pathway.
MATERIALS AND METHODS
Preparation of CSE
Kentucky 1R3F research-reference filtered cigarettes (The Tobacco Research Institute, University of Kentucky, Lexington, KY) were smoked using a peristaltic pump (VWR, Radnor, PA). The filters were cut from the cigarettes prior to the experiments. Each cigarette was smoked in 6 min with a 17 mm butt remaining. Four cigarettes were smoked through 40 mL of cell growth medium, and this solution was referred to as 100% strength CSE. The solution was adjusted to a pH of 7.45 and used within 15 min of preparation.
Cell Culture and Treatments
The human lung fibroblast cell line, MRC-5, was cultured in DMEM (Gibco-BRL, Grand Island, NY) containing 10%
FBS, in humidified incubators containing an atmosphere of 95% air, 5% CO2 at 37℃. For CSE treatments, MRC-5 cells were grown to 90% confluence and restored to fresh medium.
In addition, the cells were pre-treated with Rolipram (Sigma, St Louis, MO) for 1 hr and exposed to 20% CSE for the in- dicated time intervals.
Cytotoxicity and Viability Assays
Lactate dehydrogenase (LDH) release was measured using a cytotoxicity detection kit (Wako Pure Chemical Industries, Ltd., Tokyo, Japan), according to the manufacturer’s protocol and reagents. The reaction was initiated in 96-well plates by mixing 50 μL of cell-free supernatant with KPO4 buffer con- taining NADH and sodium pyruvate in a final volume of 100 μL. The absorbance of the sample was read at 570 nm on a mi- croplate reader (Model 680, BioRad, Hercules, CA). Data
were normalized to the activity of LDH released from gluta- mate-treated cells.
Cell viability was assessed using the (3-(4, 5-dimethylth- iazolyl-2)-2, 5-diphenyltetrazolium bromide) (MTT) assay. In brief, the formation of blue formazan was measured spec- trophotometrically from the metabolism of MTT by mi- tochondrial dehydrogenases that are active only in live cells.
MRC-5 cells were seeded in 24-well plates (5 × 104 cells/mL) and then incubated in DMEM medium for 24 hr. The cells were then pretreated with various concentrations of Rolipram for 1 hr, and then incubated with 20% CSE for an additional 24 hrs. Subsequently, MTT reagent (5 mg/mL) was added to each of the wells, and the plate was incubated for an additional 1 hr at 37℃. The media was then removed, and the intra- cellular formazan product was dissolved in 250 μL of DMSO.
The absorbance of each well was measured at 540 nm using a microplate reader (Model 680, BioRad). OD values from un- treated control cells were designated as 100% viability.
Caspase Activity Assays
Caspase-3 and -8 activities were determined by a colori- metric assay using kits from R&D Systems (Wiesbaden-Norden- stadt, Germany), according to the manufacturer’s protocols.
Briefly, cells were lysed in the manufacturer’s lysis buffer and incubated on ice for 10 min. At the end of the incubation, cell lysates were centrifuged at 10,000 × g for 10 min at 4℃ to precipitate cellular debris. The supernatants were collected and incubated at 37℃ with the supplied reaction buffer con- taining dithiothreitol and DEVD-pNA (specific for caspase-3) or IETD-pNA (specific for caspase-8) as substrates. The reaction was measured by changes in absorbance at 405 nm using an ELISA reader (BioRad, Hercules, CA). Enzyme activity was expressed as the fold increase in the proportion of apoptotic cells in treated cells relative to that of non-treated control cells.
Nuclear Staining with Hoechst 33342
Apoptosis was investigated by staining MRC-5 cells with Hoechst 33342 (Sigma, St Louis, MO). MRC-5 cells were
washed twice with PBS and then fixed in PBS containing 10% formaldehyde for 4 hr at room temperature. Fixed cells were washed with PBS and stained with Hoechst 33342 for 30 min at room temperature. Cells were evaluated under a fluorescence microscope (Olympus DP-50, Olympus Corp., Tokyo, Japan) for nuclei showing typical apoptotic features such as chromatin condensation and fragmentation. Photo- graphs were taken at a magnification of 200×.
Flow Cytometric Analysis with Annexin-V/Propidium Iodide Staining
Surface exposure of phosphatidylserine in apoptotic cells was quantitatively detected using the annexin V-FITC and propidium iodide (PI) apoptosis detection kit (Beckton Dickinson, San Jose, CA). Briefly, cells were seeded into 6-well plates (1 × 105 cells/mL) and incubated for 24 hr. After 1 hr incubation with 20% CSE at varying Rolipram con- centrations, the cells were harvested, centrifuged at 5,000 rpm, and washed twice with ice-cold PBS. Annexin V-FITC and PI double-staining was performed according to the manu- facturer’s instructions. Cell apoptosis was analysed on a FACScan flow cytometer (Beckton Dickinson, San Jose, CA).
Annexin V-FITC-positive, PI-negative cells were scored as apoptotic. Double-stained cells were scored as either necrotic or late apoptotic.
Western Immunoblot Analyses
Cells were lysed in 2 × SDS sample buffer, and protein con- centrations of the lysates were measured with the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA). Equal amounts of protein were resolved on a 4-20% sodium dodecyl sul- phate-polyacrylamide gel and transferred by electroblotting to a nitrocellulose membrane. Western blots were incubated for 2 hr with the following primary antibodies: β-actin (1:200) (Sigma); cleaved caspase-8 (1:500) and cleaved caspase-3 (1:500) (Cell Signaling). The membranes were washed with PBS containing 5% nonfat dry milk at room temperature and incubated for 1 h with horseradish peroxidase conjugated sheep anti-mouse and anti-rabbit immunoglobulin antibody
Figure 1. Cigarette smoke extract (CSE) induces MRC-5 cell death, which is inhibited by rolipram. (A) MRC-5 cells were exposed to 20% CSE for the indicated times, and cell viability was evaluated using the MTT assay. Cells treated with 20% CSE showed significant decreases in viability in a time-dependent manner relative to untreated cells. (B) Rolipram pre-treatment (10-30 μM) protects against CSE-induced cell death in MRC-5 cells. Cells were pre-treated for 1 hr with the indicated concentrations of Rolipram and then exposed to 20% CSE for 24 hr. Cell viability was evaluated using the MTT assay. (C) Rolipram protects CSE induced cell death in MRC-5 cells. Cells were exposed to 20% CSE for 24 hr after 1 hr pre-treatment with Rolipram at the indicated concentrations (10-30 μM), and cell cytotoxicity was assessed by the LDH release assay. Data from cells exposed to CSE after Rolipram pretreatment were compared to the value from cells treated with CSE alone using the Student’s t test. *p<0.05 and **p<0.01.
(1:500) (Amersham Pharmacia Biotech, Tokyo, Japan) under the same conditions. After washing with TBS-T, the specific signals were detected with an enhanced chemiluminescence detection system (Amersham Pharmacia Biotech, Uppsala, Sweden).
Statistical Analysis
All experiments were independently conducted at least 3 times. Data were expressed as the means ± S.E. Statistical dif- ferences were analyzed using the one-way ANOVA followed by Tukey's test, using the SPSS statistical software package (Version 14.0, Chicago, IL). A value of p<0.05 was consid- ered significant.
RESULTS
Rolipram Pretreatment Protects Against CSE-Induced MRC-5 Cell Death
We first investigated the effect of Rolipram on CSE-in- duced MRC-5 cell viability. MRC-5 cells were exposed to 20% CSE for 12, 24 and 48 hr, and cell viability was determined by the MTT assay. As shown in Fig. 1A, cell survival was decreased in a time-dependent manner after 24 hr exposure. Next, MRC-5 cells were pretreated for 1 hr with
Rolipram at various concentrations (10-30 μM), exposed to 20% CSE for 24 hours, and then cell viability was evaluated using the MTT assay. Rolipram pretreatment dose-depend- ently protected against the loss of cell viability induced by 20% CSE exposure (Fig. 1B).
We also evaluated the protective potential of Rolipram in this model, by evaluating cell death using the LDH release assay. MRC-5 cells were exposed to 20% CSE for 24 hr, after pretreatment with Rolipram at various concentrations (10-30 μM) (Fig. 1C). LDH release after CSE exposure was significantly decreased in Rolipram pretreated cells, in a dose-dependent manner, compared to cells treated with CSE alone.
We next evaluated morphological changes in MRC-5 cells exposed to CSE. MRC-5 cells were treated with 20% CSE for 24 hr after 1 hr pre-treatment with Rolipram at the in- dicated concentration (10-30 μM), and then stained with Hoechst 33342. An 8 hr exposure to 20% CSE caused mild chromosomal condensation and nuclear fragmentation in some cells, with a few nuclei showing intense changes (data not shown). After 24 hr exposure to CSE (20%), there was considerable condensation and fragmentation of the DNA in many cells. In Fig. 2, typical examples of vital staining for MRC-5 DNA (Hoechst 33342) are shown. The arrows in-
A B C
Figure 2. Rolipram prevents nuclear damage after CSE exposure. MRC-5 cells were pretreated with Rolipram at the indicated concentrations (10-30 μM), and then treated with 20% CSE for 24 hr. Cells were stained with Hoechst 33342 to detect morphological features of apoptosis. White arrows indicate fragmented or condensed nuclei. Photographs are representative results from three independent experiments. All panels are the same scale (magnification of 200×).
Figure 3. Rolipram prevents fibroblast apoptosis in response to CSE. (A) MRC-5 cells were pre-treated with Rolipram at the indicated concentrations (10-30 μM), and then exposed to 20% CSE for 12 hrs. (B) Flow cytometric analysis was performed using Annexin V and PI staining. Annexin V (+) PI (-) cells (RL) are considered apoptotic. The histograms represent CSE-induced apoptosis of MRC-5 cells after pre-treatment with Rolipram. Results are representative of three independent experiments. Data are expressed as the mean ±S.E.M. **p<0.01 compared with untreated controls by Student’s t test.
dicate fragmented or condensed nuclei. The number of Hoechst 33342-positive nuclei decreased with Rolipram (10-30 μM) compared with the control cell exposed to CSE alone.
Rolipram Protects Against CSE-Induced MRC-5 Cell Apoptosis
MRC-5 cells were pre-treated with Rolipram at various
concentrations and then exposed to 20% CSE for 12 hr.
Apoptosis was assessed by FACs analysis after Annexin V/PI staining. The rate of apoptosis of MRC-5 cells (Annexin V positive, PI negative cells) was dose-dependently decreased (p<0.05) by Rolipram pretreatment in cells exposed to 20%
CSE for 12 hrs, relative to that observed in cells treated with CSE alone Untreated cells served as a negative control. The cell apoptosis rates in CSE treated cells (20%, 12 h) were A
B
Figure 4. Rolipram decreases caspase-3 and -8 activities in MRC-5 cells after CSE exposure. MRC-5 cells were pre-treated with Rolipram at the indicated concentrations (10-30 μM), and then exposed to 20% CSE for the indicated time intervals. Caspase-3 (A) and caspase-8 (B) activity was determined in cell lysates. Data are expressed as the mean
±S.E.M. *p<0.05 and **p<0.01 compared with untreated controls by Student’s t test. (C) Activation of caspase-3/caspase-8 was assessed by Western blotting in total cell lysates. β-actin served as the standard.
Figure 5. Rolipram increased Akt phosphorylation in CSE- treated MRC-5 cells. (A) MRC-5 cells were exposed to 20% CSE for the indicated time points or (B) pre-in- cubated with Rolipram (30 μM) for 1 h with or without LY294002 (LY, 10 μM) and then exposed to 20% CSE for 2 hr. Total protein was isolated and subjected to Western blot analysis for p-Akt/Akt expression. (C) MRC-5 cells were pretreated for 1 hr with Rolipram (Ro) (30 μM) with or without LY294002 (LY, 10 μM), and then exposed to 20% CSE for 24 hrs. Cell viability was evaluated using the MTT assay. *p<0.05 compared with untreated controls by Student’s t test.
80.45%, 14.96%, 8.45% and 6.57% respectively, after pre-treatment with 0, 10, 20 and 30 μM of Rolipram (Fig. 3A, B). These results suggest that Rolipram protected against CSE-induced apoptotic cell death.
Rolipram Decreased Caspase-3, -8 Expression and Activity in MRC-5 Cells After CSE Exposure
MRC-5 cells were pre-treated with varying Rolipram con- centrations and then exposed to 20% CSE at the indicated time points. We evaluated the activation of caspase-8 as a function of CSE exposure, since the proteolytic auto-activa-
tion of caspase-8 activates downstream caspase-1 and cas- pase-3. Caspase-3 is an executioner caspase, whose activation represents a distal event in the apoptosis signaling pathway.
The activity of caspase-3 (Fig. 4A) or caspase-8 (Fig. 4B) was significantly (p<0.05) decreased in total cell lysates by Rolipram pre-treatment (10-30 μM) after 8 and 12 hr ex- posure to 20% CSE, compared to cells treated with CSE alone (Fig. 4A). Activation of caspases-3, or -8 was also assessed by Western immunoblotting in total cell lysates. After exposing MRC-5 cells to 20% CSE for 8 and 12 h, cell lysates were im- munoblotted with anti-caspase-3 or anti-caspase-8. There was
A B C
A B
C
a clear decrease in the appearance of the cleaved active caspase-8 p18 subunit after Rolipram pretreatment in cells exposed to CSE, compared to cells exposed to CSE alone (Fig. 4B).
Caspase-3 is normally present in an inactive pro-enzyme form, but it can be activated by proteolytic processing of its inactive zymogen into its cleaved p17 and p19 forms. A marked expression of the cleaved caspase-3 subunits p19 and p17 was evident in MRC-5 cells after 8 and 12 hr of CSE exposure. The expression of p19/p17 was decreased after pre- treatment with Rolipram (Fig. 4B).
The Protective Effects of Rolipram Against
CSE-Induced Apoptosis in MRC-5 Cells Involves Akt Phosphorylation
The Akt cascade is known to mediate cellular survival. We have previously demonstrated a protective role for Akt in CSE-induced apoptosis [17]. As shown in Fig. 5A, Akt phos- phorylation was induced by CSE treatment and sustained for 2 h post-treatment. Pretreatment with Rolipram for 1 hr showed a further increase of Akt phosphorylation in CSE treated cells.
The induction of Akt phosphorylation by Rolipram was abol- ished by treatment with the PI3K inhibitor LY294002 (10 μM) (Fig. 5B). The protective effects of Rolipram on cell viability were reversed by treatment with the PI3K inhibitor LY294002 (10 μM) (Fig. 5C).
DISCUSSION
Among the pharmacotherapeutic treatments currently available for COPD, none can reverse or prevent disease pro- gression. Inhaled bronchodilators (i.e., β2-adrenoreceptor agonists and muscarinic receptor antagonists) represent the current mainstays of COPD therapy, but primarily serve to re- lieve symptoms. The rationale for developing PDE4 in- hibitors to treat COPD is based on the ability of these com- pounds to increase intracellular cAMP levels in immune, pro-inflammatory and structural cells, in which cAMP can act as a second messenger. A wide range of cellular processes are
regulated by cAMP-dependent responses, including cellular growth, sensory signaling, neuroplasty, transcription, chemo- taxis, activation, degranulation and adherence of in- flammatory cells, as well as the release of inflammatory medi- ators (i.e, TNF-α, IL-8, and GM-CSF) [18-21]. The PDE4 in- hibitor Roflumilast can induce two cAMP-dependent signal- ing pathways: the protein kinase A (PKA)-dependent phos- phorylation of the cAMP-response element binding protein, and the Epac (exchange protein directly activated by cAMP)-dependent phosphorylation of Akt, both which confer protection against cardiomyocyte apoptosis [14]. Pre-clinical studies have demonstrated that PDE4 inhibitors consistently reduced the accumulation of neutrophils in bronchoalveolar lavage fluid following short-term exposure to CS in mice [22,23]. Thus, PDE4 inhibitors have been exploited primarily for their anti-inflammatory effects in the treatment of COPD, especially in the phenotype of chronic bronchitis.
In the present study, we investigated the effects of a PDE4 inhibitor in CSE-induced apoptosis of lung structural cells which may be implicated in the pathogenesis of emphysema.
In structural cells including lung fibroblasts, the mechanism by which PDE4 inhibitors protect against CSE-induced apop- tosis remains unclear. Several studies have examined the ef- fects of PDE4 inhibitors on emphysema [12,22]. Martorana et al. reported that Roflumilast partially ameliorated lung in- flammation and fully prevented emphysema induced by CS.
The authors suggested a role of anti-inflammatory effects of Roflumilast in the prevention of CS-induced matrix destruc- tion and emphysema [22]. The PDE4 inhibitor GPD-1116 was also shown to attenuate emphysema by inhibiting CS-induced MMP-12 activity, thereby protecting lung cells from apopto- sis [15]. Thus, these studies, extrapolated from findings ob- tained in a model of CS exposure, suggest that inflammatory events may play a central role in the development of emphyse- ma, and that neutrophil elastase activity may be a causative factor.
In our present study we show that a PDE4 inhibitor directly protected against apoptosis of lung fibroblasts in response to CSE by inhibiting caspase-3, and -8 activation. Furthermore,
we used flow cytometric analysis to demonstrate a dramatic decrease of apoptotic cells in response to CSE after Rolipram pretreatment. These findings are unique with respect to de- scribing a regulatory role of PDE4 inhibitors in the CSE-in- duced extrinsic apoptotic pathway. In our previous studies, we have shown that prevention of apoptosis inhibited CS-induced emphysema in mice [17]. We observed that CSE-induced apoptosis was differentially regulated by protein kinase C (PKC)-α and PKC-ζ via the PI3K⁄Akt pathway. We also showed that mice infected with PKC-α adenoviral vectors displayed diminished caspase-3, and -8 activities and emphy- sema in total lung tissue, relative to LacZ-infected mice [17].
It has been reported that cAMP-dependent Akt activation can inhibit apoptosis [24]. Kwak et al. reported that Akt phos- phorylation was induced by Roflumilast (30 μM) treatment and that the PI3K/Akt inhibitor LY294002 blocked the en- hanced Akt phosphorylation and the associated protective ef- fects [14]. In the present studies, Rolipram pretreatment for 1 hr caused a further increase of Akt phosphorylation in CSE-exposed MRC-5 cells. The increase in Akt phosphor- ylation and associated cytoprotection by Rolipram was dimin- ished by the PI3K inhibitor LY294002 (Fig. 5B and C). These findings suggest that the mechanism for inhibition of CSE-in- duced apoptosis by Rolipram in MRC-5 cells depended in part on the activation of the PI3K/Akt pathway.
Previously, we have shown that aminophylline, a non- specific PDE inhibitor, inhibited CSE-induced apoptosis of lung fibroblasts through the inactivation of caspases-3 and -8 [16]. Assessment of cell viability and death by MTT, LDH re- lease, and flow cytometric assays revealed that aminophylline (10 μg/mL) exerted anti-apoptotic effects against CSE-in- duced MRC-5 cell death [16]. In the present study, we have shown that Rolipram exerts an anti-apoptotic effect in a dose-dependent manner (Fig. 3), whereas aminophylline ex- erted anti-apoptotic effects at a relatively low dose [16].
In spite of their collective effects on PDE inhibition, there are many differences between the mechanisms of action of the nonselective PDE inhibitors, aminophylline or theophylline, and that of the selective PDE4 inhibitors. PDE4 inhibitors
demonstrated selective potency in PDE4 inhibition leading to increases of cAMP concentrations without action on ad- enosine receptors. In addition, second generation compounds have significant benefits over the first generation of PDE4 inhibitors.
Consistent with our previous studies, we used MRC-5 fi- broblast cells to model CSE-induced apoptotic cell death [16,17]. The alveolus is composed of a virtual syncitial net- work between alveolar type I and type II epithelial cells, endo- thelial and fibroblastic cells [25]. This structural networking provides a potential mechanism for the pathogenic dysfunc- tion that compromises all alveolar septal cells as the result of CS exposure. Alveolar destruction requires that type II and type I epithelial cells, endothelial cells, and myofibroblastic cells are destroyed concordantly in response to CS. Previous studies support the conclusion that fibroblasts represent im- portant targets for a wide variety of toxic stimuli including CS [26,27]. CS-induced apoptosis in human lung fibroblasts may contribute to the development of pulmonary emphysema in the lungs of smokers [28].
Kim et al. [29] have reported that CS induces death-induc- ing signaling complex (DISC) formation and caspase-8 acti- vation, hallmarks of the extrinsic apoptotic pathway, in human bronchial epithelial cells (Beas-2B), consistent with results observed in fibroblasts. In our previous studies, we have shown that CSE concentrations of 10-20% strongly induced DISC formation, involving the recruitment of pro-caspase-8 to the death receptor Fas [17,30]. Thus, the present study showed that 20% CSE caused lung fibroblast cell death in a manner consistent with our previous studies [16,17], and that Rolipram, a PDE4 inhibitor, protected against CSE-induced cell death. The effects of PDE4 inhibitor on CSE-induced au- tophagy are not yet defined and needed to study in the future.
In summary, we report here for the first time that the PDE4 inhibitor Rolipram protects lung fibroblasts from CSE in- duced-apoptosis via inhibiting caspases-3,-8 activation. The activation of Akt may play a contributory role in the an- ti-apoptotic role of Rolipram. The limitations of this in vitro study include the use of CSE, which provides an approx-
imation of CS exposure, but may not recapitulate all the ef- fects of mainstream CS. These findings may provide new in- sight into the mechanisms responsible for the pharmaco- logical activity of Rolipram for the treatment of CS-related lung disease such as emphysema, in addition to its known an- ti-inflammatory effects.
REFERENCES
1. Shapiro SD. The pathogenesis of emphysema: the elastase:
antielastase hypothesis 30 years later. Proc Assoc Am Physicians.
1995;107:346-52.
2. Barnes PJ, Shapiro SD, Pauwels RA. Chronic obstructive pulmonary disease: molecular and cellular mechanisms. Eur Respir J. 2003;22:672-88.
3. Sakao S, Tatsumi K, Hashimoto T, Igari H, Shino Y, Shirasawa H, et al. Vascular endothelial growth factor and the risk of smoking- related COPD. Chest. 2003;124:323-7.
4. Saetta M, Turato G, Maestrelli P, Mapp CE, Fabbri LM. Cellular and structural bases of chronic obstructive pulmonary disease.
Am J Respir Crit Care Med. 2001;163:1304-9.
5. Penning-van Beest F, van Herk-Sukel M, Gale R, Lammers JW, Herings R. Three-year dispensing patterns with long-acting inhaled drugs in COPD: a database analysis. Respir Med. 2011;
105:259-65.
6. Burge PS, Calverley PM, Jones PW, Spencer S, Anderson JA, Maslen TK. Randomised, double blind, placebo controlled study of fluticasone propionate in patients with moderate to severe chronic obstructive pulmonary disease: the ISOLDE trial. BMJ.
2000;320:1297-303.
7. Calverley PM, Boonsawat W, Cseke Z, Zhong N, Peterson S, Olsson H. Maintenance therapy with budesonide and formoterol in chronic obstructive pulmonary disease. Eur Respir J. 2003;
22:912-9.
8. Boswell-Smith V, Spina D. PDE4 inhibitors as potential therapeutic agents in the treatment of COPD-focus on roflumilast.
Int J Chron Obstruct Pulmon Dis. 2007;2:121-9.
9. Torphy TJ. Phosphodiesterase isozymes: molecular targets for novel antiasthma agents. Am J Respir Crit Care Med. 1998;
157:351-70.
10. Calverley PM, Rabe KF, Goehring UM, Kristiansen S, Fabbri LM, Martinez FJ. Roflumilast in symptomatic chronic obstructive pulmonary disease: two randomised clinical trials. Lancet. 2009;
374:685-94.
11. Fabbri LM, Calverley PM, Izquierdo-Alonso JL, Bundschuh DS, Brose M, Martinez FJ, et al. Roflumilast in moderate-to-severe chronic obstructive pulmonary disease treated with longacting
bronchodilators: two randomised clinical trials. Lancet. 2009;
374:695-703.
12. Cortijo J, Iranzo A, Milara X, Mata M, Cerda-Nicolas M, Ruiz-Sauri A, et al. Roflumilast, a phosphodiesterase 4 inhibitor, alleviates bleomycin-induced lung injury. Br J Pharmacol.
2009;156:534-44.
13. Henson PM, Vandivier RW, Douglas IS. Cell death, remodeling, and repair in chronic obstructive pulmonary disease? Proc Am Thorac Soc. 2006;3:713-7.
14. Kwak HJ, Park KM, Choi HE, Chung KS, Lim HJ, Park HY.
PDE4 inhibitor, roflumilast protects cardiomyocytes against NO-induced apoptosis via activation of PKA and Epac dual pathways. Cell Signal. 2008;20:803-14.
15. Mori H, Nose T, Ishitani K, Kasagi S, Souma S, Akiyoshi T, et al.
Phosphodiesterase 4 inhibitor GPD-1116 markedly attenuates the development of cigarette smoke-induced emphysema in senescence- accelerated mice P1 strain. Am J Physiol Lung Cell Mol Physiol.
2008;294:L196-204.
16. Kim YJ, Kim JY, Yoon JY, Kyung SY, Lee SP, Jeong SH, et al.
Protective effect of aminophylline against cigarette smoke extract-induced apoptosis in human lung fibroblasts (MRC-5 cells). Basic Clin Pharmacol Toxicol. 2011;109:17-22.
17. Park JW, Kim HP, Lee SJ, Wang X, Wang Y, Ifedigbo E, et al.
Protein kinase C alpha and zeta differentially regulate death- inducing signaling complex formation in cigarette smoke extract- induced apoptosis. J Immunol. 2008;180:4668-78.
18. Beavo JA, Brunton LL. Cyclic nucleotide research-still expanding after half a century. Nat Rev Mol Cell Biol. 2002;3:710-8.
19. Bundschuh DS, Eltze M, Barsig J, Wollin L, Hatzelmann A, Beume R. In vivo efficacy in airway disease models of roflumilast, a novel orally active PDE4 inhibitor. J Pharmacol Exp Ther. 2001;297:280-90.
20. Underwood DC, Bochnowicz S, Osborn RR, Kotzer CJ, Luttmann MA, Hay DW, et al. Antiasthmatic activity of the second- generation phosphodiesterase 4 (PDE4) inhibitor SB 207499 (Ariflo) in the guinea pig. J Pharmacol Exp Ther. 1998;287:
988-95.
21. Profita M, Chiappara G, Mirabella F, Di Giorgi R, Chimenti L, Costanzo G, et al. Effect of cilomilast (Ariflo) on TNF-alpha, IL-8, and GM-CSF release by airway cells of patients with COPD.
Thorax. 2003;58:573-9.
22. Martorana PA, Lunghi B, Lucattelli M, De Cunto G, Beume R, Lungarella G. Effect of roflumilast on inflammatory cells in the lungs of cigarette smoke-exposed mice. BMC Pulm Med. 2008;
8:17.
23. Le Quement C, Guenon I, Gillon JY, Valenca S, Cayron-Elizondo V, Lagente V, et al. The selective MMP-12 inhibitor, AS111793 reduces airway inflammation in mice exposed to cigarette smoke.
Br J Pharmacol. 2008;154:1206-15.
24. Cullen KA, McCool J, Anwer MS, Webster CR. Activation of
cAMP-guanine exchange factor confers PKA-independent protection from hepatocyte apoptosis. Am J Physiol Gastrointest Liver Physiol. 2004;287:G334-43.
25. Sirianni FE, Chu FS, Walker DC. Human alveolar wall fibroblasts directly link epithelial type 2 cells to capillary endothelium. Am J Respir Crit Care Med. 2003;168:1532-7.
26. Nakamura Y, Romberger DJ, Tate L, Ertl RF, Kawamoto M, Adachi Y, et al. Cigarette smoke inhibits lung fibroblast proli- feration and chemotaxis. Am J Respir Crit Care Med. 1995;
151:1497-503.
27. Carnevali S, Nakamura Y, Mio T, Liu X, Takigawa K, Romberger DJ, et al. Cigarette smoke extract inhibits fibroblast-mediated collagen gel contraction. Am J Physiol. 1998;274:L591-8.
28. Carnevali S, Petruzzelli S, Longoni B, Vanacore R, Barale R, Cipollini M, et al. Cigarette smoke extract induces oxidative stress and apoptosis in human lung fibroblasts. Am J Physiol Lung Cell Mol Physiol. 2003;284:L955-63.
29. Kim HP, Wang X, Chen ZH, Lee SJ, Huang MH, Wang Y, et al.
Autophagic proteins regulate cigarette smoke-induced apoptosis:
protective role of heme oxygenase-1. Autophagy. 2008;4:887-95.
30. Park JW, Yoon JY, Kim YJ, Kyung SY, Lee SP, Jeong SH, et al.
Extracellular signal-regulated kinase (ERK) inhibition attenuates cigarette smoke extract (CSE) induced-death inducing signaling complex (DISC) formation in human lung fibroblasts (MRC-5) cells. J Toxicol Sci. 2010;35:33-9.
