Infection Respiratory Epithelium During Adenoviral Stat3 Is Required

Stat3 Is Required for Cytoprotection of the Respiratory Epithelium during Adenoviral Infection

This information is current as Yohei Matsuzaki, Yan Xu, Machiko Ikegami, Valérie of September 24, 2021. Besnard, Kwon-Sik Park, William M. Hull, Susan E. Wert and Jeffrey A. Whitsett J Immunol 2006; 177:527-537; ; doi: 10.4049/jimmunol.177.1.527

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Stat3 Is Required for Cytoprotection of the Respiratory Epithelium during Adenoviral Infection1

Yohei Matsuzaki, Yan Xu, Machiko Ikegami, Vale´rie Besnard, Kwon-Sik Park, William M. Hull, Susan E. Wert, and Jeffrey A. Whitsett2

The role of Stat3 in the maintenance of pulmonary homeostasis following adenoviral-mediated lung injury was assessed in vivo. Stat3 was selectively deleted from bronchiolar and alveolar epithelial cells in Stat3⌬⌬ mice. Although lung histology and function were unaltered by deletion of Stat3 in vivo, Stat3⌬⌬ mice were highly susceptible to lung injury caused by intratracheal administration of AV1-GFP, an early (E) region 1- and E3-deleted, nonproliferative adenovirus. Severe airspace enlargement, loss of alveolar septae, and sloughing of the bronchiolar epithelium were observed in Stat3⌬⌬ mice as early as 1 day after exposure to the virus. Although surfactant protein A, B, and C content and surfactant protein-B mRNA expression in Stat3⌬⌬ mice were similar, Downloaded from TUNEL staining and caspase-3 were increased in alveolar type II epithelial cells of Stat3⌬⌬ mice after exposure to virus. RNA microarray analysis of type II epithelial cells isolated from Stat3⌬⌬ mice demonstrated significant changes in expression of numerous genes, including those genes regulating apoptosis, supporting the concept that the susceptibility of Stat3-deficient mice to adenovirus was related to the role of Stat3 in the regulation of cell survival. AV1-Bcl-xL, an E1- and E3-deleted, nonproliferative ⌬⌬ adenovirus expressing the antiapoptotic protein Bcl-xL, protected Stat3 mice from adenoviral-induced lung injury. Adenoviral infection of the lungs of Stat3-deficient mice was associated with severe injury of the alveolar and bronchiolar epithelium. Thus, http://www.jimmunol.org/ Stat3 plays a critical cytoprotective role that is required for epithelial cell survival and maintenance of alveolar structures during the early phases of pulmonary adenoviral infection. The Journal of Immunology, 2006, 177: 527–537.

ignal transducers and activators of transcription include a that Stat3 is not required for normal lung morphogenesis or func- family of structurally related proteins that play important tion but is required for maintenance of surfactant homeostasis, S roles in the intracellular transduction of signals regulated lung function, and repair following hyperoxia-induced injury (6). by various cytokines and growth factors. Stat3 was initially iden- Although IL-6, IL-11, and activated Stat3 protect the lung during tified as a transcription factor that mediated the effects of IL-6 in oxidant injury in vivo (7–9), mechanisms by which activation of acute phase response in the liver (1). Stat3 mediates or participates Stat3 maintains lung homeostasis following injury are poorly by guest on September 24, 2021 in the signaling pathways of many cytokines (e.g., IL-6, IL-11, understood. IL-10, IL-2, leukemic inhibitory factor, ciliary neurotrophic factor, The recent severe acute respiratory syndrome outbreak (10) and oncostatin M, leptin, and others) and growth factors (e.g., epider- the ongoing morbidity and mortality associated with influenza and ␣ mal growth factor, TGF- , hepatocyte growth factor, and G-CSF) other pulmonary viral infections has served to raise awareness of in various cells and organs (1–3). Cytokines and growth factors the public health consequences of viral pneumonias and the need activate Stat3 via glycoprotein 130 (gp130)-activating phosphor- for further knowledge regarding the pathogenesis of pulmonary ylation by JAKs. Phosphoryl-Stat3 dimerizes and is transported to viral infections. The mechanisms protecting the respiratory epithe- the nucleus, where it regulates the transcription of target genes. lium from acute cellular injury after exposure to respiratory viruses Because systemic deletion of Stat3 in transgenic mice is lethal at E6.5-7.5 (4), the biological roles of Stat3 have been determined in are complex and include induction of antioxidant, antiapoptotic vitro and after conditional deletion of the gene in various cell types pathways, specific enhancement of cell proliferation, and surfac- and organs of the mouse. Stat3 plays a critical role in the regulation tant production (8, 11, 12). Activation of Stat3 and pre-exposure to of various biological processes, including cell survival, apoptosis, IL-6 protects the lung from injury during hyperoxia (7, 9). inflammation, and proliferation (5). Conditional ablation of Stat3 The lung is repeatedly subjected to injury caused by viral in- in the respiratory epithelium of mice (Stat3⌬⌬ mice) demonstrated fection and other pathogens. Maintenance of pulmonary homeostasis requires the continuation of cellular processes and proliferation of cells during viral infection. Infected cells are cleared by acute Division of Pulmonary Biology, Cincinnati Children’s Hospital Medical Center, De- partment of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH and immune-mediated apoptosis and necrosis. In this study, we 45229 assessed the role of Stat3 in pulmonary homeostasis during adeno- Received for publication December 13, 2005. Accepted for publication April viral-mediated lung injury. A conditional system was used to ex- 10, 2006. press Cre-recombinase, selectively deleting the Stat3 gene in bron- The costs of publication of this article were defrayed in part by the payment of page chiolar and alveolar epithelial cells of the mouse lung (6). charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Intratracheal administration of AV1-GFP, an E1- and E3-deleted, 1 This work was supported by National Institutes of Health Grants HL61646 (to M. nonproliferative adenovirus, caused severe lung pathology associ- Ikegami, S.E. Wert, and J.A. Whitsett) and HL38859 (to J.A. Whitsett). ated with enhanced apoptosis of the respiratory epithelium in 2 Address correspondence and reprint requests to Dr. Jeffrey A. Whitsett, Cincinnati Stat3⌬⌬ mice. These results demonstrate that Stat3 plays a critical Children’s Hospital Medical Center, Divisions of Neonatology and Pulmonary Biol- ogy, 3333 Burnet Avenue, Cincinnati, OH 45229-3039. E-mail address: role in cytoprotection of the lung during the early phases of viral [email protected] pneumonia.

Table I. Comparison of histology scoring in control and Stat3⌬/⌬a

Saline AV1-GFP Statstical Analysis

Control vs Stat3⌬/⌬ Stat3⌬/⌬ ϩ Saline vs Morphometric Analysis Control Stat3⌬/⌬ Control Stat3⌬/⌬ after AV1-GFP Stat3⌬/⌬ ϩ AV1-GFP

Alveolar epithelium Mean Mean Mean Mean p Value p Value Airspace enlargement 0.1 0.3 0.3 3.6 Ͻ0.05 Ͻ0.05 Loss of alveolar septae 0.1 0.3 0.1 3.5 Ͻ0.05 Ͻ0.05 Congestion 0.2 0.1 1.4 4.1 Ͻ0.05 Ͻ0.05 Hemorrhage 1.0 1.1 1.1 1.5 N.S. Ͻ0.05 Alveolar protein 0.9 0.9 1.3 2.9 Ͻ0.05 Ͻ0.05 Bronchiolar epithelium Epithelial sloughing 0.0 0.2 0.4 1.9 Ͻ0.05 Ͻ0.05 Cellular necrosis 0.0 0.0 0.0 0.0 NS NS Inflammation Neutrophil infiltrates 0.0 0.0 0.1 0.5 NS NS Macrophage infiltrates 1.2 0.8 1.7 3.6 Ͻ0.05 Ͻ0.05

a Histology from four mice per group was analyzed 48 h after exposure to AV1-GFP or saline. Fourteen lobes for saline exposure groups, 15 lobes for AV1-GFP exposure groups. Lobes were scored from 0 to 5 with 0 being normal or no histopathology, and 5 being the most severely affected. Kruskal-Wallis one-way analysis and Dunn’s method were performed with individual scores for each characteristic to determine signiﬁcant difference; signiﬁcance was accepted at the 5% level. Downloaded from

Materials and Methods resentative of ﬁndings from at least four Stat3⌬⌬ mice compared with Gene construction and doxycycline administration control mice.

flx/flx SP-C-rtTA/(tetO)7CMV-Cre/Stat3 triple-transgenic mice were gener- TUNEL assay flx/flx ated as described previously (6). Stat3 mice were a gift from Dr. http://www.jimmunol.org/ Takeda (Hyogo College of Medicine, Hyogo, Japan) (4, 13). In the pres- The TUNEL assay was performed on tissue paraffin sections and cryosec- ence of doxycycline, exon 21 of Stat3 gene was permanently deleted from tions using In Situ Cell Death Detection Kit (Roche) according to the respiratory epithelial cells before birth (Stat3⌬⌬ mice) (6). To confirm ge- manufacturer’s protocol. notype, DNA was purified from tail or lung of experimental mice, and PCR flx Morphometric analysis was performed for SP-C-rtTA, (tetO)7CMV-Cre, and Stat3 genes using Ј Ј the following primers: 5 -GAC ACA TAT AAG ACC CTG GCTA-3 and Severity of adenoviral-induced lung injury was assessed using a system Ј Ј 3 5 -AAA ATC TTG CCA GCT TTC CC-3 for surfactant protein (SP) -C- that scored alveolar epithelial injury, bronchiolar injury, inflammation, and rtTA; 5Ј-TGC CAC GAC CAA GTG ACA GCA ATG-3Ј and 5Ј-AGA ⌬⌬ Ј Ј alveolar size (Table I). Lung sections from multiple lobes of Stat3 and GAC GGA AAT CCA TCG CTCG-3 for (tetO)7CMV-Cre; and 5 -CCT control mice (14–15 lobes/n ϭ 4 per group) were assessed following saline GAA GAC CAA GTT CAT CTG TGT GAC-3Ј and 5Ј-CAC ACA AGC

or AV1-GFP exposure by a blinded reviewer. Kruskal-Wallis one-way by guest on September 24, 2021 CAT CAA ACT CTG GTC TCC-3Ј for Stat3flx. Stat3-deleted transgenic ⌬⌬ flx/flx analysis and Dunn’s method were performed with individual scores for (Stat3 ) and nondeleted littermates (Stat3 , control mice) were used each characteristic to determine significant differences. Significance was for the experiments. Doxycycline was administered to the dams in the food accepted at the 5% level. at a concentration of 625 mg/kg (Harlan Teklad) from embryonic day 0 (E0) to postnatal day 25 (P25), resulting in extensive deletion of Stat3 in Quantification of apoptosis and cleaved caspase-3-reactive cells respiratory epithelial cells. Mice were then provided normal food. Body ⌬⌬ weights of adult control and Stat3 mice were similar, 24.75 Ϯ 3.0g(n ϭ To quantify the TUNEL reaction and cleaved caspase-3 signals, 10 random ⌬⌬ 89) for control and 24.69 Ϯ 3.0g(n ϭ 86) for Stat3 mice. fields per animal were photographed by fluorescence microscopy. Selection of each field was decided by a randomization function of Microsoft Excel. Intratracheal administration of adenovirus Apoptosis signals were counted manually, and 4Ј,6Ј-diamidino-2-phenylin- dole signals were counted by thresholding, using MetaMorph software AV1-GFP, AV1-Bcl-xL, and AV1-Bcl-2, all E1- and E3-deleted, nonproliferative type 5 adenoviruses sharing an identical viral background, were (Molecular Devices). The ratio of apoptosis-positive cells:total cell number was calculated, and reported as the apoptosis cell index (mean Ϯ SEM). generated in 293 cells (14, 15). AV1-Bcl-xL and AV1-Bcl-2 were a gift from Dr. Molkentin (Cincinnati Children’s Hospital, Cincinnati, OH) (16). For apoptosis analysis, comparisons among groups were made by ANOVA Adenovirus was diluted with HBSS (Invitrogen Life Technologies) or PBS with Tukey’s test used for post hoc analysis. Results were expressed as to 2 ϫ 1010 optical particle unit (opu)/mouse and administered intratra- mean and SEM. Significance was accepted at the 5% level. cheally in a total volume of 80 ␮l (12). Adenovirus or saline (experimental control) were administered to 8- to 10-wk-old Stat3⌬⌬ and control mice, by Western blot analysis for SPs intubation with 24-gauge animal feeding needles (Popper & Sons) and a The content of SP-A, SP-B, and SP-C in bronchoalveolar lavage fluid laryngoscope (Bio Research Center) during anesthesia with isoflurane. (BALF) was estimated by Western blot analysis. Samples were obtained by Tissue preparation, histology, and immunohistochemistry (IHC) pooling three 1-ml lung lavage washes. Recovery volumes were similar in each group of animals. BALF (30 ␮l) for SP-B, and SP-A and SP-C (10 ␮l) Lung tissues were prepared as described previously (17). IHC was per- were diluted in Laemmli buffer and subjected to SDS-PAGE under non- formed essentially as described previously (18). Tissue sections were reducing conditions for SP-B (19) and under reducing conditions for SP-A stained with H&E. Primary Abs were used at the following dilutions: SP-B and SP-C (20). The following dilutions of Ab were used: SP-A (1/5000, (1/2,000, rabbit polyclonal; Chemicon International), pro-SP-C (1/4,000, guinea pig polyclonal; Chemicon International), SP-B (1/5000 rabbit poly- rabbit polyclonal; Chemicon International), Stat3 (1/400, rabbit polyclonal; clonal; Chemicon International), and SP-C (1/5000, rabbit polyclonal). Per- Cell Signaling), phosphoryl-Stat3 (Tyr705) (1/50, rabbit polyclonal; Cell oxidase-conjugated secondary Abs (Calbiochem, EMD Biosciences) were Signaling), and cleaved caspase-3 (the active form of caspase-3, 1/500 for used at 1/5000 dilutions. Immunoreactive bands were detected with ECL immunofluorescence, rabbit polyclonal; R&D Systems). Immunofluores- reagents (Amersham Health). cent, secondary Ab Alexa Flour 568-tagged goat anti-rabbit IgG was used at a dilution of 1/200 (Molecular Probes). All experiments shown are rep- RNA extraction and RNA analysis for SPs Lung tissues were excised and homogenized with TRIzol reagent (Invitro- 3 Abbreviations used in this paper: SP, surfactant protein; opu, optical particle unit; gen Life Technologies). RNA concentration was measured by spectropho- IHC, immunohistochemistry; BALF, bronchoalveolar lavage fluid; APP, amyloid ␤ tometer. S1 nuclease assays for SP-B, SP-C, and L32 mRNA were per- 4A precursor protein. formed as described previously (21). The Journal of Immunology 529

Isolation alveolar type II epithelial cells Alveolar type II cells were isolated from 6-wk-old control and Stat3⌬⌬ mice 2 h after isolation using collagenase and differential plating as described by Rice et al. (22). Type II cells were used for FACS and type II cell culture.

Apoptosis analysis following AV1-GFP exposure in vitro Isolated type II cells were counted with a hemacytometer. Equivalent numbers of cells were cultured on 100% Matrigel. After 4 days of culture on the gel, cells were exposed to saline or AV1-GFP (2 ϫ 107 PFU/well, 2 ϫ 108 PFU/well, 2 ϫ 109 PFU/well). Twenty-four hours after exposure, cells were removed from the Matrigel with Cell Recovery Solution (BD Bio- sciences). The recovered type II cells were rinsed with cold PBS and cen- trifuged. The cell pellets were resuspended in 50 ␮l of Laemmli buffer and sonicated. Samples (20 ␮l) were analyzed by Western blot of cleaved caspase-3 (1/1,000, rabbit polyclonal; Cell Signaling). ␤-Actin (1/10,000, mouse polyclonal; Seven Hills Bioreagents) was used as a control. Perox- idase-conjugated secondary Abs (Calbiochem, EMD Biosciences) were used at 1/2,000 for cleaved caspase-3 and 1/10,000 for actin.

RNA microarray analysis Downloaded from mRNA was extracted from pools of alveolar type II epithelial cells isolated from Stat3⌬⌬ and control mice. Each group had three males and three females, 6 wk of age. The cRNA was hybridized to the murine genome MOE430 chip (consists of Ϸ45,000 gene entries) (Affymetrix) according to the manufacturer’s protocol. Affymetrix Microarray Suite 5.0 was used to scan and quantitate the gene chips under default scan setting. Normal- ization was performed using the Robust Multichip Average model (23, 24). http://www.jimmunol.org/ Data were further analyzed using Linear Models for Microarray Data (25). Differentially expressed genes were selected with the threshold of Stu- dent’s t test ( p Ͻ 0.05; false discovery rate Ͻ10% and fold change Ͼ1.5). Unknown genes/expressed sequence tags and duplicated gene entries were temperately removed from further functional analysis. Genes associated with apoptosis were identified by PubMed Browser. Gene Ontology (GO) analysis was performed using public available web-based tool DAVID (database for annotation, visualization and integrated discovery) (26). FIGURE 1. Lung histology following pulmonary adenoviral infection (day 2). Histology was assessed with H&E staining 2 days after exposure Validation of mRNAs of 8-wk-old control (A, C, E, and G) and Stat3⌬⌬ (B, D, F, and H) mice to by guest on September 24, 2021 AV1-GFP. Control and Stat3⌬⌬ mice were administered 2 ϫ 1010 opu of Real-time RT-PCR was used to cross-validate changes in several mRNAs AV1-GFP or saline. Severe airspace enlargement, loss of alveolar septae, detected by IHC and microarray analysis. Malt-1-1, rtn4, reg3g, and Bcl-xL ⌬⌬ were detected using primers listed (see Table IV). Changes in mRNA were and sloughing of airway epithelium were observed in Stat3 mice fol- ⌬⌬ Ն determined in type II cells isolated from Stat3 and controls (n ϭ 3–4/ lowing AV1-GFP exposure (D and H). Figures are representative of n 5 group). Student t test was performed. Significance was accepted at the 5% per genotype. Scale bar, 100 ␮m. level. The following primers were used: Malt1: forward, TAT CCA GGA GGA CCC CAT GT and reverse, TCT GAT CAA AGC CAG TTA GCA TCAT; Rtn4: forward, AAG TGG AAG GAG TTT GAG AGA GCA and ⌬⌬ reverse, CTG TCT CAA AGC AGA TGT GAA AGC; Reg3g: forward, SP expression was maintained in Stat3 mice TGC CAA AAG AGC CCT CAG GA and reverse, TGC CTG AGG AAG The concentration of SPs (SP-A, SP-B, and SP-C) was analyzed in AGG AAG GAT TCG; Bcl-xL: forward, TCT CTC TCC TCT GTC CAC CCT TG and reverse, TGC CCC TCA GAA GCC AGA AC. BALF 48 h after administration of adenovirus (Fig. 4A). In both Stat3⌬⌬ and control mice, the levels of SPs in BALF increased following adenovirus administration. SP content in BALF was not Results ⌬⌬ ⌬⌬ different in Stat3 and control mice after exposure to the virus, at Stat3 mice were susceptible to adenoviral-mediated lung injury times during which severe lung injury was observed in the Stat3⌬⌬ Lung histology was assessed 1–7 days following intratracheal ad- mice. Thus, the early susceptibility of the Stat3⌬⌬ mice to lung ministration of AV1-GFP. Severe airspace enlargement, loss of injury was not likely mediated by changes in SP expression. SP alveolar septae, and sloughing of the bronchiolar epithelium were mRNAs in whole lung were analyzed by S1 nuclease assay (Fig. observed in lungs of Stat3⌬⌬ mice as early as 1 day after exposure 4B). Levels of SP mRNAs were not altered by infection or deletion to the virus (Figs. 1 and 2 and Table I). In contrast, airspace ab- of Stat3, consistent with the lack of change in SP levels in BALF. normalities were not observed in control mice, consistent with pre- IHC for SP-B and pro-SP-C was performed 48 h after intratra- vious studies with this virus (18, 27). Severe airspace enlargement cheal administration of adenovirus. Although SP staining in the related to loss of alveoli, bronchiolar sloughing, and focal hemor- alveoli was not different in Stat3⌬⌬ mice, SP-B and pro-SP-C- rhage were observed 1, 2, 5, and 7 days after infection in Stat3⌬⌬ stained cells were fewer, likely representing the generalized loss of mice. In contrast, lung inflammation was substantially resolved in alveolar tissue following adenovirus (Fig. 5). Because there was no control mice during the same time period. Thus, Stat3⌬⌬ mice were difference in the levels of SP in BALF after adenoviral exposure, more susceptible to lung injury and alveolar simplification follow- we hypothesized that lung injury and alveolar loss observed in ing adenoviral infection. Consistent with the importance of Stat3 Stat3⌬⌬ mice was not mediated by changes in extracellular SP during the early phase of infection, activation of Stat3 was indi- homeostasis. Airspace enlargement and alveolar loss was observed cated by nuclear staining of phospho-Stat3 observed 30 min after in the Stat3⌬⌬ mice rather than atelectasis, the latter generally as- adenoviral infection in control mice (Fig. 3). sociated with surfactant deficiency. 530 Stat3 AND CYTOPROTECTION OF THE LUNG Downloaded from

FIGURE 3. Immunohistochemical staining of phosphoryltyrosine Stat3 following adenoviral infection of control mice. Lung tissues were inﬂation ﬁxed 30 min (A), 60 min (B), 120 min (C), and 1 day (D) following in- http://www.jimmunol.org/ tratracheal administration of 2 ϫ 1010 opu of AV1-GFP, and 1 day (E) following saline. Tissue was immunostained with phosphoryl-Stat3 (Tyr705). Stat3 activation was observed in the conducting airways 30 min after adenovirus administration and in alveolar epithelial cells within 60 min. Scale bar, 100 ␮m.

FIGURE 2. Lung histology following pulmonary adenoviral infection (day 7). Lung sections were prepared 7 days after exposure of control (A, Stat3 regulates expression of genes modulating apoptosis and C, E, and G) and Stat3⌬⌬ (B, D, F, and H) mice to 2 ϫ 1010 opu of by guest on September 24, 2021 other cellular processes AV1-GFP or saline and stained with H&E. More airspace loss was observed in Stat3⌬⌬ mice following AV1-GFP. Figures are representative of The mRNA expression profiles were compared in type II epithelial n Ն 5 per genotype. Scale bar, 100 ␮m. cells isolated from Stat3⌬⌬ and control mice using Affymetrix murine genome MOE430 gene chips. mRNAs derived from n ϭ 1425 genes were identified as significantly altered using the criteria described in Materials and Methods. Expression of 887 mRNAs was Apoptosis increased in Stat3⌬⌬ mice following adenoviral increased while that of 538 mRNAs was decreased in response to infection the deletion of Stat3. Differentially expressed genes were further Cleaved caspase-3 IHC and TUNEL assay was used to determine whether epithelial cell apoptosis might mediate the extensive loss of the alveolar epithelium seen in the Stat3⌬⌬ mice following adenoviral infection. The TUNEL assay was performed on lung tissue from Stat3⌬⌬ and control mice 24 h after exposure to AV1- GFP or saline (Fig. 6). Consistent with this observation, cleaved caspase-3-positive cells were increased in lung epithelial cells of Stat3⌬⌬ mice following adenovirus exposure (Fig. 6). The number of caspase-3 and TUNEL-positive cells was significantly increased in Stat3⌬⌬ mice following AV1-GFP compared with controls (Fig. 7).

Increased caspase-3 activity following AV1-GFP exposure in type II cells from Stat3⌬⌬ mice in vitro To further assess whether Stat3 plays a role in protection of the FIGURE 4. SP expression and SP-B mRNA were not altered by dele- epithelial cells from apoptosis, primary alveolar type II cells from tion of Stat3. A, SP-A, SP-B, and SP-C were quantitated in BALF by ϫ 10 Stat3⌬⌬ and control mice were isolated and treated with AV1-GFP Western blot analysis 48 h after intratracheal instillation of 2 10 opu of AV1-GFP. AV1-GFP increased SPs in BALF of both Stat3⌬⌬ and con- ϫ 7 Ϫ ϫ 9 (2 10 2 10 PFU/well) or saline. Changes in caspase-3 trol mice. Deletion of Stat3 did not alter SP content following treatment were analyzed by Western blot analysis (Fig. 8). Cleaved with AV1-GFP. B, SP-B and SP-C mRNAs were quantitated by S1 nucle- caspase-3 was signiﬁcantly increased in type II cells isolated from ase assay. SP-B and SP-C mRNAs were similar in control and Stat3⌬⌬ ⌬⌬ Stat3 mice 24 h after exposure to the adenovirus in vitro. mice following adenoviral exposure. The Journal of Immunology 531 Downloaded from

FIGURE 7. Quantiﬁcation of TUNEL and cleaved caspase-3. TUNEL assay was assessed in lung sections from mice receiving 2 ϫ 1010 opu of AV1-GFP. The proportion of TUNEL-positive cells in Stat3⌬⌬ mice fol- http://www.jimmunol.org/ lowing AV1-GFP was increased signiﬁcantly. Statistical differences were .(p Ͻ 0.05 ,ء) assessed by ANOVA test

classified according to GO classification on Biological Process us- FIGURE 5. IHC for SP-B and pro-SP-C. SP-B (A–D) and pro-SP-C ing public available web-based tool DAVID (26). The Fisher exact (E–H) immunohistochemical staining was performed in control and test was used to calculate the probability that category was over- Stat3⌬⌬ mice 48 h after exposure to 2 ϫ 1010 opu of AV1-GFP (D). In- ⌬⌬ represented using the entire MOE430 mouse genome as reference tracellular staining for SP-B was slightly decreased in Stat3 mice fol- ϭ by guest on September 24, 2021 lowing AV1-GFP. SP-B was detected in the alveolar spaces and in type II dataset (Table II). Genes regulating protein transport ( p 8.30E- ⌬⌬ ϭ epithelial cells in the Stat3 mice following AV1-GFP. Cellular staining 12), transcription ( p 4.41E-07), and programmed cell death/ for pro-SP-C (E–H)inStat3⌬⌬ and control mice was used to identify type apoptosis ( p ϭ 8.59E-07) were significantly increased after dele- II epithelial cells. Photomicrographs are representative of n Ն 4. Scale bar, tion of Stat3, while lipid, carboxylic acid, and organic acid 100 ␮m. metabolism (with p values of 8.34E-07, 3.5E-07, and 3.54E-07, respectively) were selectively decreased by deletion of Stat3. Thus, despite the lack of observable abnormalities in the lungs of Stat3⌬⌬ mice before challenge with virus, expression of numerous genes was altered by the deletion of Stat3. Of particular interest, a number of genes regulating cell survival and apoptosis were influenced by deletion of Stat3 in type II epithelial cells (Table III). Selected mRNAs identified by microarray analysis including Malt-

1-1, Rtn4, Reg3a, and Bcl-xL were assessed by real-time RT-PCR, demonstrating similar changes in expression of a number of genes inﬂuencing cell survival (Table IV).

Bcl-xL prevents adenoviral-mediated lung injury in vivo TUNEL-positive cells and cleaved caspase-3 were increased in Stat3⌬⌬ mice following exposure to AV1-GFP, indicating that

FIGURE 6. Increased cleaved caspase-3 and TUNEL staining in FIGURE 8. Increased cleaved caspase-3 in alveolar type II cells from Stat3⌬⌬ mice. Immunostaining for cleaved caspase-3 and the TUNEL assay Stat3⌬⌬. Western blotting of cleaved caspase-3 was performed in primary were performed 24 h after intratracheal administration of 2 ϫ 1010 opu of type II cells 24 h after saline (a), AV1-GFP 2 ϫ 107 PFU/well (b), 2 ϫ 108 AV1-GFP in vivo. Caspase-3 (red, arrow) and TUNEL (green) were in- PFU/well (c), and 2 ϫ 109 PFU/well (d) in vitro. Cleaved caspase-3 was creased in Stat3⌬⌬ mice following adenoviral infection. Photomicrographs increased in type II cells from Stat3⌬⌬ after AV1-GFP exposure was in- are representative of n Ն 5. Scale bar, 100 ␮m. creased relative to dose of AV1-GFP. 532 Stat3 AND CYTOPROTECTION OF THE LUNG

Table II. Functional classification of categories of genes influenced by sloughing of airway epithelial cells seen after AV1-GFP exposure deletion of Stat3 in type II epithelial cellsa were not observed following AV1-Bcl-xL administration. Expres- sion of Bcl-xL in 293 cells was increased 24 h after exposure to Functional Classification of Up-Regulated Genes AV1-Bcl-xL, demonstrating the activity of the virus in vitro. The GO Term Percentage p Value TUNEL assay was performed on lung tissue from Stat3⌬⌬ and

Physiological process 44.9 6.14E-21 control mice 24 h after exposure to AV1-GFP, AV1-Bcl-xL,or Cellular physiological process 21.1 1.74E-13 saline. Although TUNEL signals increased following AV1-GFP Metabolism 30.3 2.87E-13 exposure, TUNEL signals after AV1-Bcl-xL were similar to that Regulation of biological process 13.8 7.19E-12 seen after saline exposure (Fig. 10). In contrast, treatment of the Protein transport 4.1 8.30E-12 ⌬⌬ Protein metabolism 13.6 1.72E-11 Stat3 mice with the same titer of AV1-Bcl-2 did not ameliorate Intracellular transport 4.6 1.79E-11 viral-induced injury in vivo (data not shown). Macromolecule metabolism 15.7 1.86E-11 Intracellular protein transport 3.8 7.08E-11 Protein modiﬁcation 6.8 3.03E-10 Discussion ⌬⌬ Intracellular signaling cascade 5.7 8.16E-10 Stat3 mice were highly susceptible to AV1-GFP Regulation of metabolism 10.2 1.61E-07 Although respiratory epithelial cell-speciﬁc deletion of Stat3 did Regulation of physiological process 10.4 2.10E-07 ⌬⌬ Cell death 3 3.53E-07 not alter lung morphogenesis or function (6), Stat3 mice were Transcription 9.9 4.41E-07 highly susceptible to lung injury and apoptosis and alveolar loss Death 3 6.48E-07

following intratracheal administration of a nonproliferative E1- Downloaded from Transcription, DNA-dependent 9.4 6.98E-07 and E3-deleted adenovirus, AV1-GFP. Histological studies dem- Programmed cell death 2.8 8.59E-07 Apoptosis 2.7 1.17E-06 onstrated remarkable airspace enlargement, rapid loss of alveolar Regulation of nucleobase, nucleoside, 9.4 1.69E-06 septae, sloughing of the respiratory epithelium, and increased in- nucleotide and nucleic acid flammation in the Stat3⌬⌬ mice. Increased TUNEL staining and metabolism enhancement of cleaved caspase-3 expression were observed after Regulation of transcription 9.3 4.64E-06 ⌬⌬ exposure of Stat3 mice to the replication-defective adenovirus. Regulation of transcription, DNA- 9 5.92E-06 http://www.jimmunol.org/ dependent Thus, Stat3 is critical for cytoprotection of the respiratory epithe- Nucleobase, nucleoside, nucleotide 13 1.59E-05 lium following adenoviral infection. Although Stat3 plays an es- and nucleic acid metabolism sential role in maintenance of pulmonary surfactant homeostasis Transcription from POL II promoter 1.7 5.27E-05 during oxygen-induced injury (6–9), the early and severe epithe- Phosphate metabolism 4.4 6.31E-05 ⌬⌬ Phosphorus metabolism 4.4 6.31E-05 lial cell injury seen following adenoviral infection in Stat3 mice Negative regulation of nucleobase, 1 6.90E-04 was not associated with decreased SPs in the airspace. The present nucleoside, nucleotide and nucleic findings support that Stat3 is required for survival of the respira- acid metabolism tory epithelium during early phases of pulmonary viral infection. Regulation of programmed cell death 1.5 1.00E-03 by guest on September 24, 2021 Negative regulation of transcription 1 1.18E-03 ⌬⌬ Regulation of apoptosis 1.5 1.80E-03 Severe lung injury seen in Stat3 mice was not due to Negative regulation of metabolism 1 4.62E-03 surfactant deficiency or dysfunction Cholesterol metabolism 0.4 4.45E-02 Because Stat3 is known to regulate expression of SP-B (Sftpb) and Functional Classification of Down-Regulated Genes to be critical for the maintenance of lung function during hyper- GO Term Percentage p Value oxia (6, 9, 32), we assessed SPs content in BALF after adenoviral exposure in the Stat3⌬⌬ mice. SP content in lung lavage fluid was Metabolism 31.4 1.25E-11 ⌬⌬ Lipid biosynthesis 2 4.31E-09 similar in Stat3 and control mice following adenovirus admin- Physiological process 44.3 1.10E-08 istration. Likewise, SP-B and SP-C mRNAs were not influenced Lipid metabolism 3.4 8.34E-07 by Stat3 deletion or AV1-GFP exposure, 24 and 48 h after expo- Macromolecule metabolism 14.8 1.46E-05 sure to the virus. However, the number of cells and the intracel- Fatty acid biosynthesis 0.6 2.13E-03 lular staining for SP-B and pro-SP-C were slightly decreased in Carboxylic acid biosynthesis 0.6 2.22E-03 ⌬⌬ Organic acid biosynthesis 0.6 2.22E-03 Stat3 mice 24 and 48 h after adenoviral exposure, at times when Catabolism 4.7 1.07E-02 lung injury and airspace enlargement were extensive. The decrease Secretion 0.6 1.13E-02 in intracellular immunohistochemical staining for SP-B and pro- Microtubule-based process 1.1 1.31E-02 SP-C may represent changes in intracellular content, including a GO analysis was performed using a publicly available web-based tool DAVID. synthesis, processing, routing, or secretion that may be directly or The Fisher exact test was used to calculate the probability of each category that was overrepresented in the selected list using the entire MOE430 mouse genome as the indirectly influenced by Stat3, or represent that numbers of stained reference dataset. cells per residual alveoli appeared decreased, because the virus caused extensive loss of alveolar cells per se. The finding that SP-B and SP-C mRNAs were unchanged suggest that the change

Stat3-deﬁcient cells may be susceptible to apoptosis. Bcl-xL and in staining is not mediated by direct effects of Stat3 on their tran- Bcl-2, Bcl-2 family members, are known to inhibit apoptosis (28– scription. Because the half-life of SP-B protein is ϳ6–8hinthe

31). To assess whether Bcl-xL or Bcl-2 prevents adenovirus-in- adult mouse lung (33), the observed differences in intracellular duced apoptosis, AV1-Bcl-xL and AV1-Bcl-2, E1- and E3-deleted, SP-B staining were not reflected by changes in alveolar SP-B con- nonproliferative adenoviruses, of identical genetic background to centrations. Pathologic findings in the lung of AV1-GFP-infected AV1-GFP, was administered intratracheally to Stat3⌬⌬ and control mice were not consistent with findings associated with surfactant mice. Lung histology was assessed 2 days after infection with deficiency or dysfunction that are generally indicated by atelectasis

AV1-Bcl-xL and AV1-Bcl-2. Lung histology was substantially im- and edema hemorrhage. In contrast to the present ﬁndings, in- proved in mice receiving AV1-Bcl-xL compared with AV1-GFP creased alveolar hemorrhage and atelectasis were observed during (Figs. 9 and 10). Airspace enlargement, loss of alveolar septae, and 4–5 days of hyperoxia that were mediated primarily by surfactant The Journal of Immunology 533

Table III. mRNA expression proﬁles were compared in type II epithelial cells 2 h after isolation from Stat3⌬/⌬ and control micea

Genes Protecting from Apoptosis Gene Ratio Gene title

Akt2 Ϫ1.55 Thymoma viral proto-oncogene 2 Atf5 Ϫ1.59 Activating transcription factor 5 Ϫ Bcl-211 1.54 Bcl2-like 1 (Bcl-xL) Nme5 Ϫ2.04 Expressed in nonmetastatic cells 5 Prdx2 Ϫ1.57 Peroxiredoxin 2 Reg3g Ϫ5.92 Regenerating islet-derived 3␥

Api5 2.63 Apoptosis inhibitor 5 Bcl-2 1.52 B cell leukemia/lymphoma 2 Bcl-2a1a 2.39 Bcl-2-related protein A1a Birc3 1.51 Baculoviral IAP repeat-containing 3 Birc4 3.26 Baculoviral IAP repeat-containing 4 Birc6 2.39 Baculoviral IAP repeat-containing 6 Bnip2 1.89 Bcl-2/adenovirus E1B 19 kDa-interacting protein 1 Bfar 1.87 Bifunctional apoptosis regulator Pik3rl 2.42 Phosphatidylinositol 3-kinase, regulatory subunit polypeptide 1 Mcl1 1.65 Myeloid cell leukemia sequence 1 Igf1 2.25 Insulin-like growth factor 1 Downloaded from Tnfaip3 2.16 TNF␣ induced protein 3 Tnfrsf11b 2.33 TNF receptor superfamily member 11b Traf1 2.88 TNF receptor-associated factor 1

Genes Inducing Apoptosis Gene Ratio Gene title http://www.jimmunol.org/ App 1.52 Amyloid precursor protein Bcl10 1.97 Bcl-2-like 10 Bcl2l11 2.26 Bcl-2-like 11 Bclaf1 3.49 Bcl-2-associated transcription factor 1 Bnip31 1.99 Bcl-2/adenovirus E1B 19kDa-interacting protein 3-like Bcap29 1.51 B cell Rc-associated protein 29 Casp3 2.02 Caspase 3 Cox7c 2.15 Cytochrome C oxidase, subunit VIIc Cul1 1.79 Cullin 1 Faf1 1.69 Fas-associated factor 1 by guest on September 24, 2021 Gzmb 2.21 Granzyme B Pik3c2a 3.15 Phosphatidylinositol 3-kinase, C2 domain containing␣polyp Malt1 7.66 Mucosa associated lymphoid tissue lymphoma translocation gene 1 Plag11 2.09 Pleiomorphic adenoma gene-like 1 Pten 2.32 Phosphatase and tensin homolog Rad21 2.33 RAD21 homolog Rtn4 5.59 Reticulon 4 Ripk1 1.72 Receptor (TNFRSF)-interacting serine-threonine kinase 1 Sgpp1 1.75 Sphingosine-1-phosphate phosphatase 1 Sh3glb1 1.67 SH3-domain GRB2-like B1 Stk17b 4.29 Serine/threonine kinase 17b Tde1 2.95 Tumor differentially expressed 1 Tnfsf6 2.72 TNF superfamily, member 6 Trp53inp1 1.75 Trp 53-inducible nuclear protein 1

Amid Ϫ1.59 AIF-like mitochondrion-associated inducer of death Bad Ϫ1.54 Bcl-associated death promoter Bak1 Ϫ1.75 Bcl-2-antagonist/killer 1 Bbc3 Ϫ1.65 Bcl-2 binding component 3 Bcap31 Ϫ1.65 B cell Rc-associated protein 31 Bcl2l14 Ϫ1.66 Bcl-2-like 14 (apoptosis facilitator) Biklk Ϫ2.19 Bcl-2-interacting killer-like Bmf Ϫ1.63 Bcl-2 modifying factor Cidea Ϫ2.47 Cell death-inducing DNA fragmentation factor, ␣ subunit- like Dedd Ϫ1.56 Death effector domain-containing E2f2 Ϫ3.01 E2F transcription factor 2 Eya2 Ϫ2.01 Eyes absent 2 homolog Hras1 Ϫ2.07 Harvey rat sarcoma virus oncogene 1 Mkl1 Ϫ1.66 MKL-like 1 Mtch1 Ϫ1.79 Mitochondrial carrier homolog 1 Siva Ϫ1.76 Cd27 binding protein Sphk2 Ϫ1.74 Sphingosine kinase 2 Traf4 Ϫ1.72 TNF receptor associated factor 4 Trib3 Ϫ1.68 Tribbles homolog 3 Vdac1 Ϫ1.58 Voltage-dependent anion channel 1

a Genes associated with apoptosis were identiﬁed by PubMed Browser and GO analysis using Pathway Assist. Stat3 regulated a group of genes associated with cell survival/apoptotic pathways. Ratio is expression in Stat3⌬/⌬ vs control. IAP, interacting protein. 534 Stat3 AND CYTOPROTECTION OF THE LUNG

Table IV. Comparison of mRNAs by RT-PCR and RNA microarraya

Ratio Stat3⌬/⌬ to Control

Gene RT-PCR Microarray Affymetrix ID p Value for RT-PCR

Malt1-1 4.5 7.66 1456429 Ͻ0.05 Rtn4 3.6 6.46 1442760 Ͻ0.05

Reg3g Ϫ5.5 Ϫ5.92 1448872 Ͻ0.05 Ϫ Ϫ Bcl-xL 1.2 1.54 1420888 0.053 a Ratio of mRNA in Stat3⌬/⌬ mice to control is shown. deﬁciency and decreased SP-B (6). The severe alveolar loss pres- ently observed after virus exposure was not observed in the Stat3⌬⌬ mice during oxygen exposure, supporting the requirement for Stat3 for prevention of alveolar injury after adenoviral exposure was distinct from its role in the regulation of surfactant ho-

meostasis during hyperoxia. Downloaded from ⌬⌬ FIGURE 10. Bcl-xL reduces TUNEL staining. TUNEL assay was per- TUNEL staining was increased in epithelial cells in Stat3 formed 24 h after intratracheal instillation of saline (A), AV1-GFP (B), or mice following exposure to adenovirus. Variability in TUNEL AV1-Bcl-xL (C). TUNEL-positive cells were increased following AV1- staining was observed and that may represent variability of the GFP in Stat3⌬⌬ mice (B). TUNEL signal was similar to that of saline- ⌬⌬ Stat3 gene targeting, in the distribution of the virus after intratra- treated mice (A) following AV1-Bcl-xL to Stat3 mice (C). Photomicro- cheal administration or efﬁcacy of clearance of apoptotic cells graphs are representative of n Ն 5. Scale bar, 100 ␮m.

from the lung. Immunohistochemical staining for cleaved http://www.jimmunol.org/ caspase-3 and increased caspase activity were observed in Stat3- deficient mice and in type II cells isolated from Stat3⌬⌬ mice. Type II cells, identified by pro-SP-C staining, represented 85% of the ceptibility of the epithelial cells rather than macrophages or fibro- cells isolated from adult mouse lungs by flow cytometry. Because, blasts. This study demonstrates that viral-induced apoptosis was in this model, deletion of Stat3 occurs only in epithelial cells, controlled by Stat3 in the respiratory epithelium in a cell-autono- increased caspase-3 activation likely indicates the increased sus- mous manner.

Mechanism of adenoviral-induced apoptosis

When infected with adenovirus, cells express viral proteins on the by guest on September 24, 2021 cell surface mediated by MHC class 1 TCR. CTLs recognize the exposed viral proteins causing apoptosis via granzyme or FAS ligand-mediated pathways. Activation of CTL generally occurs 4–7 days after viral infection (34). In this study, however, apoptosis was observed 1–2 days after adenoviral exposure, before the activation of CTL-mediated apoptosis. These ﬁndings support the likelihood that adenoviral proteins per se induce apoptosis in the absence of Stat3. Although E1a protein of the adenovirus is an initiator of apoptosis (35–37), the E1 region is deleted in the AV1- GFP construct (14). Adenovirus also induces apoptosis via the pro-death gene product, E4ORF4 (38, 39), although the mechanisms by which this protein is unknown at present. Although E4ORF4 is intact in the AV1-GFP construct and is likely expressed following infection with this adenoviral vector, the mechanism by which AV1-GFP induces epithelial cell loss in the Stat3⌬⌬ mice remains unclear.

Role of Stat3 during adenoviral infection The present finding demonstrates that Stat3 plays a critical role in protection of the lung from an early apoptotic effect of the virus, thus avoiding catastrophic loss of lung structure. In this study, phospho-Stat3 staining increased in epithelial cells within 30 min ⌬⌬ FIGURE 9. Bcl-xL protects Stat3 mice from pulmonary adenoviral after exposure to adenovirus (40–42), indicating that Stat3 is infection. Lung histology was assessed after intratracheal administration of likely to play an important role in cytoprotection early in the pro- saline (A and B), 2 ϫ 1010 opu of AV1-GFP (C and D), or 2 ϫ 1010 opu ⌬⌬ cess of infection, perhaps limiting injury by sustaining cell survival of AV1-Bcl-xL (E and F) in control (A, C, and E) and Stat3 (B, D, and F) mice. Severe enlargement of airspace, loss of alveolar septae, and until repair processes are initiated. The observed cytoprotective sloughing of the bronchiolar epithelium were observed in Stat3⌬⌬ mice effect of Stat3 is consistent with previous findings demonstrating following AV1-GFP (C and D). Lung injury was ameliorated by expression that loss of Stat3 in macrophages and neutrophils renders cells or of Bcl-xL in the same adenoviral vector (E and F). Photomicrographs are mice susceptible to endotoxin administration (43), impairs prolif- representative of n Ն 5. Scale bar, 100 ␮m. erative response in T cell lymphocytes (13, 44), delays wound The Journal of Immunology 535 healing in keratinocytes (45), and enables susceptibility to injury, failure of survival in hepatocytes (46), cell death in motoneurons (47), and hypersensitivity of thymic epithelial cell to apoptosis (48). Stat3 regulates expression of genes regulating cell survival RNA microarray analysis demonstrated that there were many genes regulating apoptosis/cell survival that were altered after deletion of Stat3 in respiratory epithelial cells, in both positive and negative directions, and at multiple levels (Table II). Interestingly, RNAs that play an important role in proapoptotic pathways, including caspase-3, amyloid ␤ 4A precursor protein (APP), and FIGURE 11. Model of apoptotic pathways influenced by Stat3. A sim- Cox7c, were increased, perhaps consistent with the observed in- plified model is proposed by which the lack of Stat-3 influences the ex- creased sensitivity of the Stat3⌬⌬ mice to viral-induced cell loss. pression of genes that regulate cell survival. The relationship between Stat3 and genes regulating apoptosis/ cell survival were complicated (Table III). Deletion of Stat3 altered type II cells resulted in changes in mRNAs influencing mul- components of the cell survival pathways that maintains cellular tiple components mediating cell death processes including Bcl-2 homeostasis following viral infection (Fig. 11). family members (Bcl-2, Bcl211, Mc11, Bcl2111, Bak1, and Downloaded from Bcl-x protects the Stat3⌬⌬ mice from adenoviral-induced injury BAD), caspase-3, caspase-8 and Fas-associated death domain pro- L tein-like apoptosis regulator (Cflar, also known as Flip), voltage- Rapid loss of alveolar-bronchiolar epithelial cells, increased dependent anion channel 1 (Vdac1), and cytochrome C (Cox7c). TUNEL, and cleaved caspase-3 following treatment with adeno- Taken together, these findings support the concept that Stat3 in- virus strongly supported the concept that susceptibility to apopto- ⌬⌬ fluences cell survival at multiple levels (i.e., direct transcriptional sis played a role in severe injury observed in the Stat3 mice.

regulation or indirectly through the regulation of other transcrip- Likewise, mRNA array studies support the likelihood that diverse http://www.jimmunol.org/ tion factors or posttranslational modiﬁcations of other proteins). changes in expression of genes inﬂuencing cell survival were per-

Because our experimental data demonstrated susceptibility to ap- turbed by deletion of Stat3. Expression of Bcl-xL in the same viral optosis, we focused on the analysis of cell survival pathways (Ta- vector conferred cytoprotection following adenoviral infection, ble II). Stat3 is known to serve in an antiapoptotic role by enhanc- supporting the concept that deletion of Stat3 increased suscepti- ing transcription of Bcl-2 and Bcl-xL (49–54), Bcl-xL being a bility to cell death. Expression of Bcl-xL in Av1-Bcl-xL, but not ⌬⌬ direct transcription target of Stat3 (52). The decreased expression AV1-Bcl-2, protected the Stat3 mice from viral-induced lung of Bcl-xL in Stat3-deficient cells may represent a potential direct injury. Although Bcl-xL mRNA was also decreased in type II ep- transcriptional effect of the deletion of Stat3. Increased Bcl-2 and ithelial cells as assessed by the mRNA array data, many mRNAs Bcl-2a1a mRNA may indicate compensatory responses serving to influencing cell survival were altered in Stat3-deficient cells before by guest on September 24, 2021 protect cells from apoptosis. Stat3 induces apoptosis by regulating viral infection. Thus, the observed cytoprotection by AV1-Bcl-xL PI3K-Akt-mediated survival signaling (52, 55). The PI3K regula- strongly supports the important role of Stat3 in cytoprotection, but tory subunits, p55␣ and p50␣, are known as targets of Stat3 (55, does not indicate that changes in Bcl-xL alone account for the 56). PI3K controls the activity of Akt by regulating its location and observations. AV1-Bcl-2 did not protect the lung as did Bcl-xL. activation (57), Akt, in contrast, inhibits STAT3 transcriptional Bcl-2 has both antiapoptotic activity and proapoptotic activities at activity and phosphorylation (58). Several PI3K subunits were in- different concentration (65). It is unclear whether Bcl-2 is less creased, whereas Akt2 was reduced, following deletion of Stat3 as active than Bcl-xL or that Bcl-2 was not expressed at appropriate indicated by the microarray data that is likely to represent a com- levels to influence cytoprotection in our experiments. pensatory response to the lack of Stat3. The PI3K-Akt pathway In summary, the present study demonstrates that Stat3 plays a influences a wide spectrum of downstream targets that influence critical role in the regulation of a number of genes in the respira- apoptosis (58). For example, Akt phosphorylates multiple Bcl-2 tory epithelium that support cell survival during the early phases of family members including BAD and Bcl-xL (59), blocks cyto- adenoviral infection of the lung. chrome C release from mitochondria (60), and inhibits caspase-3 activation (61). Components of the apoptotic machinery interact at Acknowledgments multiple levels. Bcl-xL interacts with Vdac1 to regulate this outer We thank Ann Maher for secretarial assistance. We also thank K. Takeda mitochondrial membrane channel opening and membrane potential for the gift of Stat3flx/flx mice, B. C. Trapnell for the gift of AV1-GFP, and that controls production of reactive oxygen species and release of J. D. Molkentin for the gift of AV1-Bcl-xL and AV1-Bcl-2. cytochrome C, both of which are the potent inducers of cell apoptosis (62, 63). In the present analysis, cleaved caspase-3 staining Disclosures and caspase-3 mRNA were increased. Caspase-3 mediates cleav- The authors have no financial conflict of interest. age of APP (64). APP mRNA was also increased after deletion of Stat3. References Taken together, the present findings suggest that lack of Stat3 1. Akira, S. 1997. IL-6-regulated transcription factors. Int. J. Biochem. Cell Biol. 29: 1401–1418. causes complex changes in expression of both pro- and antiapop- 2. Darnell, J. E., Jr. 1997. STATs and gene regulation. Science 277: 1630–1635. totic proteins that regulate balance of pro- and antiapoptotic ac- 3. Boccaccio, C., M. Ando, L. Tamagnone, A. Bardelli, P. Michieli, C. Battistini, tivities that determine cell survival. Because changes in gene ex- and P. M. Comoglio. 1998. Induction of epithelial tubules by growth factor HGF depends on the STAT pathway. Nature 391: 285–288. pression represent the net outcome of complicated balances, it is 4. Takeda, K., K. Noguchi, W. Shi, T. Tanaka, M. Matsumoto, N. Yoshida, unlikely that changes in a single or even a few genes determines T. Kishimoto, and S. Akira. 1997. Targeted disruption of the mouse Stat3 gene leads to early embryonic lethality. Proc. Natl. Acad. Sci. USA 94: 3801–3804. the susceptibility of type cells to viral-induced injury. Rather, Stat3 5. Bromberg, J., and J. E. Darnell, Jr. 2000. The role of STATs in transcriptional plays a remarkable and critical role in the regulation of multiple control and their impact on cellular function. Oncogene 19: 2468–2473. 536 Stat3 AND CYTOPROTECTION OF THE LUNG

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