special topic - Lung Cellular and Molecular Physiology

Am J Physiol Lung Cell Mol Physiol 282: L370–L378, 2002; 10.1152/ajplung.00200.2001.

special topic Pre- and Postnatal Lung Development, Maturation, and Plasticity MEK-1/2 inhibition reduces branching morphogenesis and causes mesenchymal cell apoptosis in fetal rat lungs DAVID E. KLING,1 HANS K. LORENZO,1 ALEXANDER M. TRBOVICH,1 T. BERNARD KINANE,2 PATRICIA K. DONAHOE,1 AND JAY J. SCHNITZER1 1 Pediatric Surgical Research Laboratories, Pediatric Surgical Services, and the Department of Surgery; and 2Laboratory of Developmental Immunology and the Department of Pediatrics; Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114 Received 4 June 2001; accepted in final form 13 August 2001

Kling, David E., Hans K. Lorenzo, Alexander M. Trbovich, T. Bernard Kinane, Patricia K. Donahoe, and Jay J. Schnitzer. MEK-1/2 inhibition reduces branching morphogenesis and causes mesenchymal cell apoptosis in fetal rat lungs. Am J Physiol Lung Cell Mol Physiol 282: L370–L378, 2002; 10.1152/ajplung.00200.2001.—The roles of the mitogenactivated protein (MAP) kinases extracellular signal-regulated kinases-1 and -2 (ERK-1/2) in fetal lung development have not been extensively characterized. To determine if ERK-1/2 signaling plays a role in fetal lung branching morphogenesis, U-0126, an inhibitor of the upstream kinase MAP ERK kinase (MEK), was added to fetal lung explants in vitro. Morphometry as measured by branching, area, perimeter, and complexity were significantly reduced in U-0126-treated lungs. At the same time, U-0126 treatment reduced ERK-1/2, slightly increased p38 kinase, but did not change c-Jun NH2-terminal kinase activities, indicating that U-0126 specifically inhibited the ERK-1/2 enzymes. These changes were associated with increased apoptosis as measured by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and immunofluorescent labeling of anti-active caspase-3 in the mesenchyme of explants after U-0126 treatment compared with the control. Mitosis characterized by immunolocalization of proliferating cell nuclear antigen was found predominantly in the epithelium and was reduced in U-0126-treated explants. Thus U-0126 causes specific inhibition of ERK-1/2 signaling, diminished branching morphogenesis, characterized by increased mesenchymal apoptosis, and decreased epithelial proliferation in fetal lung explants.

THE PROCESSES OF LUNG GROWTH and development are extremely complex, involving a multitude of effectors including growth factors, extracellular matrix interactions, and hormones (9, 38). Many of these effectors

activate signaling pathways that converge into the mitogen-activated protein (MAP) kinases. There are three major families of MAP kinases: the extracellular signal-regulated kinases-1 and -2 (ERK-1/2), c-Jun NH2-terminal kinases (JNK), and p38 kinases. Both ERK-1 and -2 are thought to be involved primarily in proliferation and differentiation, whereas JNK and p38 are believed to be involved in stress responses and apoptosis (10). ERK-1 and -2 are 44- and 42-kDa proteins, respectively, that become activated when phosphorylated on both the tyrosine and threonine residues within the TEY motif by MAP ERK kinases (MEK)-1 and -2 (10). MEK-specific inhibitors PD-98059 and 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U-0126) have been shown to reduce ERK-1/2 signaling, with the latter having a lower IC50 than the former (8, 11). U-0126 is an organic compound that noncompetitively inhibits the catalytic activity of the active enzyme (7). Evidence for the role of ERK-1/2 signaling in lung development is circumstantial and is based on the effects of receptor tyrosine kinase (RTK) activation, including hepatocyte growth factor (HGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF) (25, 26, 28, 29, 33), and G protein activation (19). However, the signaling pathways that lead to changes in lung morphology or markers that induce differentiation have not been elucidated. The canonical model of RTK signaling requires ligand binding to its receptor, which produces a transphosphorylation reaction that is transduced through several intermediates, including Ras, and ultimately through a cassette consisting of Raf/MEK/ERK-1/2. Activated ERK-1/2 is then transported to the nucleus to induce

Address for reprint requests and other correspondence: J. J. Schnitzer, Pediatric Surgical Services, Massachusetts General Hospital, 55 Fruit St., WRN 1159, Boston, MA (E-mail: schnitzer.jay @mgh.harvard.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

lung development; mitogen-activated protein kinase; extracellular signal-regulated kinase; U-0126

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MEK INHIBITION SUPPRESSES FETAL LUNG GROWTH

gene expression (24). However, other MAP kinases including ERK-5 and its upstream and downstream partners may represent an alternative signaling pathway (16). Apoptosis, a programmed cell death characterized by membrane blebbing, activation of caspases (cysteine proteases usually inactive as zymogens), and DNA fragmentation, is induced by mitochondria-derived effectors, including cytochrome c and apoptosis-inducing factor (AIF), and by the cellular concentration of the Bcl (B-cell leukemia) family of proteins (2, 12, 22, 34). Caspase activation is hierarchical, i.e., upstream caspases (caspase-2, -8, -9, and -10) activate executioner caspases (caspase-3, -6, and -7) that carry out the destruction of the cell (32). Caspase-3 activation occurs by proteolytic cleavage of the 32-kDa proenzyme into large (17 kDa) and small (12 kDa) peptides that associate to form an active enzyme and has been used as a marker for cells undergoing apoptosis (5). Active caspase-3 cleaves downstream targets, including the DNA fragmentation factor 45 (DFF45)/inhibitor of caspase-activated DNase (ICAD), vimentin, gelsolin, and lamin B, which have also been used as markers for the occurrence of apoptosis in dying cells (32). Apoptosis has been observed to take place at several stages of lung development, indicating that it plays an important role in the remodeling that characterizes the maturation of this organ (4, 6, 21). At embryonic days 14–16 (E14–E16) in the rat, apoptosis is observed in mesenchymal cells at the periphery of distal lung buds, suggesting that apoptosis is necessary for distal branching (21). At postnatal day 17, numbers of apoptotic fibroblasts increased sharply, which correlated with the completion of alveolarization and the thinning of connective tissue (4). In rabbits, type II epithelial cell apoptosis increased significantly on E28 (term is ⬃E31), corresponding to the transition from canalicular to the terminal sac stage of development (6). This transition correlates with a 20-fold increase in FasL (a type II transmembrane protein belonging to the tumor necrosis family of ligands) mRNA immunolocalized to alveolar type II cells and bronchiolar Clara cells, indicating a role of Fas/FasL in fetal lung epithelial apoptosis (6). Thus the observed bursts of apoptosis appear to correspond to milestones in lung remodeling. Proliferating cell nuclear antigen (PCNA), also known as cyclin, has been used as a marker for cell proliferation in developing lung (18, 37). PCNA is a 36-kDa auxiliary protein of DNA polymerase-delta and accumulates during the S phase (3). During DNA synthesis a fraction of the polymerase becomes tightly associated with DNA replication sites while the remainder of the population is nucleoplasmic and is present during quiescence. Fixation of cells with methanol but not paraformaldehyde distinguishes between the replication site-bound and the nucleoplasmic populations (3). Immunolocalization of PCNA is age dependent in the lung; its occurrence in the mesenchyme is rare by midgestation and is essentially absent during the second half of pregnancy, whereas 25% of AJP-Lung Cell Mol Physiol • VOL

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epithelial cells were immunopositive for PCNA throughout early and midgestation (14). The following study examines the consequences of inhibition of MEK by U-0126 on fetal lung explants. Our data suggest that U-0126 treatment reduces lung branching morphogenesis and is associated with increased apoptosis in the mesenchyme and decreased proliferation of the epithelium. Delineation of the signaling pathways involved in lung development may ultimately lead to strategies to rescue pulmonary hypoplasia associated with a broad spectrum of diseases, including congenital diaphragmatic hernia. METHODS

Chemicals and reagents. All chemicals, reagents, and materials were purchased from Fisher Scientific (Pittsburgh, PA) unless otherwise noted. Antibiotics and amphotericin B were purchased from Sigma (St. Louis, MO). The MEK inhibitor U-0126 was purchased from Promega Biotech (Madison, WI). Antibodies to diphosphorylated ERK-1/2 (dp-ERK, E10 monoclonal), JNK (dp-JNK), p38 (dp-p38), and positive [UV-treated human embryonic kidney (HEK)-293 cell extract] and negative (untreated HEK-293 cell extract) controls for dp-p38 and dp-JNK were purchased from New England Biolabs (Beverly, MA). Other polyclonal antibodies specific for total ERK-2 (K-23), p38, JNK-1, and the PCNA monoclonal antibody were obtained from Santa Cruz Biotech (Santa Cruz, CA). Purified rabbit anti-active caspase-3 monoclonal antibody was purchased from BD PharMingen (San Diego, CA). The caspase inhibitor Z-Val-Ala-Asp(OCH3)-fluoromethylketone (zVAD-FMK) was purchased from Biomol (Plymouth Meeting, PA). Timed-pregnant rats were purchased from Zivic Miller (Pittsburgh, PA). BGJb growth medium was purchased from GIBCO (Rockville, MD). Lung explant culture. All protocols were carefully reviewed and approved by the Massachusetts General Hospital Subcommittee on Research Animal Care (accession no. 1999N001169) following the guidelines of the National Institutes of Health. Fetal lung explant culture procedures were based on methods previously described (15, 36). Pregnant dams at E13 were euthanized in carbon dioxide, and embryos were removed by cesarean section. Lungs removed from the pups were separated from the surrounding tissue using a dissecting microscope under sterile conditions and were placed over 2 ml of BGJb medium with 5,000 U/ml penicillin, 5 mg/ml streptomycin, 10 mg/ml neomycin, 2.5 ␮g/ml amphotericin B, and 0.2 mg/ml ascorbic acid (JT Baker, Phillipsburg, NJ) in Costar Transwell plates (no. 3452, Corning, NY). Stock solutions of U-0126 (20 mM) were prepared in DMSO and added to BGJb to give a final concentration of 20 ␮M. A stock solution of zVAD-FMK (50 mM) was prepared in DMSO and diluted 1/1,000 in the media. DMSO was brought to a final dilution of 1/500 in all samples. Media were changed daily. Lung explants were visualized live and unstained through the Transwell plates using a Nikon microscope (⫻25; Tokyo, Japan) and photographed with a Spot digital camera (Diagnostic Instruments, St. Sterling Heights, MI). The images were exported into Photoshop (Adobe Systems, San Jose, CA), the endoderm was traced with an Intuos graphics tablet (Wacom), and the tracings were exported into NIH Image (version 1.61/PPC) for morphometric analyses of lung bud count, area, and perimeter. Complexity was then calculated as the perimeter divided by the square root of area (perimeter/公area). 282 • MARCH 2002 •

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Statistics. Statistical analysis was carried out using the Student’s t-test with statistical significance established at P ⬍ 0.05 (30). Western blot analysis. Lysates of pooled lung explants were suspended in ice-cold lysis buffer consisting of 20 mM HEPES, pH 7.5, 50 mM ␤-glycerophosphate, 10% glycerol, 2 mM EGTA, 1% Triton X-100, 1 mM sodium vanadate, and the Complete protease inhibitor cocktail (Roche, Indianapolis, IN). The explants were homogenized with a pellet pestle (Kontes Glass, Vineland, NJ) on ice, centrifuged in a microcentrifuge for 5 min at 4°C, and transferred to a fresh tube. After protein concentrations were determined by the method of Bradford (1), 5 ␮g of protein in 6⫻ SDS sample buffer was loaded onto 10% acrylamide minigels, electrophoresed at 15 mA at room temperature until the dye front reached the bottom of the gel (1), and then transferred to Immobilon (Millipore, Bedford, MA). Blots probed with antibodies to the diphosphorylated forms of ERK, JNK, and p38 proteins were blocked in 1% BSA and 0.5% dried milk dissolved in Tris-buffered salineTween 20 (TBS-T: 50 mM Tris 䡠 HCl, pH 7.6, 150 mM NaCl, and 0.1% Tween 20) for 30 min, followed by the addition of dp-ERK, dp-p38, or dp-JNK diluted 1/2,000 and incubation overnight at 4°C. Blots were washed four times in TBS-T, incubated with a secondary horseradish peroxidase (HRP) conjugate (Jackson ImmunoResearch Laboratories, West Grove, PA) diluted 1/25,000 in blocking buffer, washed four more times in TBS-T, developed with ECL-plus (Amersham Pharmacia Biotech, Piscataway, NJ), and exposed to Biomax ML film (Kodak, Rochester, NY). Blots to be reprobed with ERK-2-, JNK-1-, or p38-specific antibodies were stripped in 62.5 mM Tris 䡠 HCl, pH 6.8, 2% SDS, and 100 mM 2-mercaptoethanol at 50°C for 30 min and washed six times with TBS-T. Blots were blocked in 5% serum (obtained from the animal used for raising the secondary antibody conjugate; Jackson ImmunoResearch Laboratories) dissolved in TBS-T for 1 h at room temperature, followed by two washes in TBS-T and hybridization with primary antibodies diluted in TBS-T plus 1% BSA for 1 h. After four washes with TBS-T, blots were incubated with HRP secondary conjugates (Jackson ImmunoResearch Laboratories) diluted 1/25,000 in TBS-T plus 1% BSA, washed four times in TBS-T, developed with ECL-plus, and exposed to Biomax ML film. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. Lungs grown in vitro were prepared for the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay by fixing in 4% paraformaldehyde for 4 h at 4°C followed by washes in 7% sucrose in PBS. The fixed tissue was then transferred first to Tissue Freezing Medium (Triangle Biomedical Sciences, Durham, NC) for 5 min, placed in Cryomolds (Miles, Elkhart, IN) filled with Tissue Freezing Medium, and then frozen in liquid nitrogen, and stored at ⫺70°C. Sections of 6 ␮m were cut with a Minotome cryostat (Damon/IEC Division, Needham Heights, MA), and sections were air dried and stored at 4°C. The TUNEL assay was carried out using an in situ cell death detection kit fluorescein (Roche), followed by a counterstain in 0.5 ␮g/ml propidium iodide (Sigma) for 5 min, and the sections were viewed using a Nikon microscope. Images were taken with a Spot digital camera, exported into Adobe Photoshop, and imported into Canvas version 7.1 (Deneba Systems, Miami, FL). Immunofluorescence. To detect active caspase-3, fetal lung explants were fixed and sectioned as described above for the TUNEL assay. Air-dried sections were permeabilized with 0.1% SDS in PBS, washed three times, and blocked with 5% AJP-Lung Cell Mol Physiol • VOL

goat serum (Jackson Immunolaboratories) for 45 min. Antiactive caspase-3 antibody was added at a dilution of 1/1,000 in PBS containing 2% BSA (PBS/2% BSA) at 4°C in a humidified chamber overnight while experimental negative controls were incubated in PBS/2% BSA without the active caspase-3 antibody. After three washes in PBS, the secondary antibody (Cy3 goat anti-rabbit, Jackson Immunolaboratories) was added at a dilution of 1/200 for 1 h. After three washes with PBS, sections were incubated with Hoechst33342 trihydrochloride trihydrate (Molecular Probes, Eugene, OR), diluted 1/20,000 in PBS to stain the nuclei, and washed three more times in PBS. Finally, the sections were analyzed with a Nikon microscope at ⫻125; images were photographed with a Spot digital camera, exported into Photoshop, and imported into Canvas. To detect PCNA, explants were fixed in 100% methanol for 30 min to overnight at ⫺20°C and desiccated in a SpeedVac Concentrator (Savant, Farmingdale, NY). The dehydrated lungs were resuspended in 100% methanol and washed in decreasing concentrations of methanol (75, 50, 25, and 0%) in Hanks’ balanced salt solution. Explants were bathed in Tissue Freezing Medium for 5 min, placed in Cryomolds filled with Tissue Freezing Medium, frozen in liquid nitrogen, and stored at ⫺70°C. Cryosections of 6 ␮m were cut, air-dried, washed once in PBS, and blocked in PBS containing 4% donkey serum (Jackson Immunolaboratories) for 1 h. After two washes in PBS, sections were incubated overnight with a solution containing PCNA-monoclonal antibody at a dilution of 1/1,000 in 2% BSA/PBS at 4°C in a humidified chamber while negative controls were incubated in 2% BSA/PBS without the PCNA antibody. These incubations were followed by three washes in PBS after which the secondary conjugate (Cy3 donkey anti-mouse antibody, Jackson Immunolaboratories) was added at a dilution of 1/200 for 1 h. Sections were washed three times with PBS, counterstained with Hoechst33342, and analyzed as described above for anti-active caspase-3. RESULTS

MEK inhibition alters lung morphology. To determine if the ERK MAP kinases play a role in lung development, fetal lung explants were treated with or without the MEK inhibitor U-0126, and the lung morphometry was characterized. After 4 days in culture [U-0126, n ⫽ 10; control (DMSO), n ⫽ 9], reduced branching morphogenesis was observed (Fig. 1, A and B) and quantified (Fig. 1, C–F) by lung bud count (Fig. 1C), area (Fig. 1D), perimeter (Fig. 1E), and complexity (perimeter/公area; Fig. 1F). All of these parameters of lung growth were significantly reduced compared with the control, suggesting that inhibition of MEK activity profoundly reduced branching morphogenesis. U-0126 specifically decreases ERK-1/2 MAP kinase activities. To verify that U-0126 specifically inhibited ERK-1/2 and not other MAP kinases, Western blot analysis with antibodies to the diphosphorylated forms of ERK-1/2 (dp-ERK-1/2), p38 (dp-p38), and JNK (dpJNK) were carried out. Figure 2A shows reduced intensity of dp-ERK-1/2 in the lanes corresponding to the U-0126-treated explants. This reduction reflected ERK-1/2 phosphorylation because there was no change in ERK-1/2 protein levels (Fig. 2B). When similarly analyzed, explants treated with U-0126 have increased levels of dp-p38 (Fig. 2C) compared with controls 282 • MARCH 2002 •

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Fig. 1. Effect of mitogen-activated protein (MAP) kinase extracellular signalregulated kinase (ERK) kinase (MEK) inhibition on lung morphometry. A: digital photomicrographs of lung explants grown in vitro for 4 days with the MEK-specific inhibitor U-0126 or without U-0126 (control, DMSO). B: epithelial outlines of representative lungs grown with U-0126 and DMSO. Quantitative analysis of lungs grown in culture: total bud count (C), area (D), perimeter (E), and complexity (perimeter/公area; F). Error bars show SD. * P ⬍ 0.001, ** P ⬍ 0.0001.

(DMSO), while p38 protein remains constant (Fig. 2D). Figure 2, E and F, shows that dp-JNK-1 (46-kDa) and JNK-1 levels are similar between the U-0126-treated explants and the DMSO-treated controls. The slight signal detected for dp-JNK likely reflects low activity of this kinase because this antibody readily detects activated JNK in a lysate of UV-treated HEK-293 cells used as a positive control (Fig. 2E). Thus U-0126AJP-Lung Cell Mol Physiol • VOL

mediated MEK inhibition does not reduce the activity or expression of either JNK-1 or p38, indicating that U-0126 specifically inhibits ERK-1/2 phosphorylation in fetal lung explants. U-0126 treatment increases apoptosis in the mesenchyme of fetal lung explants. A TUNEL assay was used to determine if apoptosis played a role in the U-0126mediated inhibition of fetal lung branching. Figure 3 282 • MARCH 2002 •

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Fig. 2. MAP kinase activities in lung explants after U-0126 treatment. Western blot analysis of MAP kinase activities in U-0126treated lung explants with antibodies to the diphosphorylated (activated) forms of ERK-1/2 (dp-ERK-1/2) (A), p38 (dp-p38; C), and c-Jun NH2-terminal kinase (JNK)-1 (dp-JNK-1; E), or protein levels of ERK-1 and ERK-2 (B), p38 (D), and JNK-1 (F) are shown. ERK-2 antibodies cross-react with the ERK-1 protein. Positive (⫹, UV-treated human embryonic kidney (HEK)-293 cells) and negative (⫺, untreated HEK-293 cells) controls were included where indicated 54 and 46 kDa.

shows that DNA fragmentation is substantially increased in the U-0126-treated explants compared with the control (DMSO). The majority of the observed cell death was localized to the mesenchyme (Fig. 3B), suggesting that these cells are more sensitive to ERK-1/2 inhibition than the epithelial cells. When the pancaspase inhibitor zVAD-FMK was used to determine if the U-0126-mediated increased cell death was caspase dependent, DNA fragmentation was reduced (Fig. 3, B and E), implying that the observed apoptosis was caspase dependent. To confirm that U-0126 induces mesenchymal cell apoptosis, activated caspase-3 was analyzed by immunofluorescence. Cryosections of fetal lung explants treated with either U-0126 or DMSO were probed with an anti-active caspase-3 monoclonal antibody (5). Figure 4 shows an increase in active caspase-3 predominantly in the mesenchyme of the U-0126-treated explant, supporting the previous observation that apoptosis occurs predominantly in the mesenchyme after U-0126 treatment. Effects of U-0126 on cell proliferation in fetal lung explants. To determine if proliferation is altered in lungs treated with U-0126, PCNA expression was examined by immunofluorescence. Figure 5A shows that PCNA is detected primarily in the epithelium in DMSO-treated (control) explants. However, the number of PCNA-positive cells is reduced in the U-0126treated explants (Fig. 5B), suggesting that U-0126 treatment reduces epithelial cell proliferation in fetal lung explants. DISCUSSION

Although several RTK cascades, including FGF, EGF, HGF, IGF, and PDGF, are known to play roles in AJP-Lung Cell Mol Physiol • VOL

pulmonary branching morphogenesis, transduction through the MAP kinases ERK-1/2 has not been demonstrated (25, 26, 28, 29, 33). Use of the MEK inhibitor U-0126 reduced ERK-1/2 signaling, which correlated with a potent inhibition of lung branching morphogenesis in vitro. This implies an important role for ERK1/2 activation in mediating lung growth and development. MAP kinase signaling is similarly activated during development in a wide variety of organisms (13, 17, 35). In Caenorhabditis elegans, for example, the ERK-1/2 homolog MPK-1 phosphorylates the wingedhelix transcription factor Lin-31, which disrupts the Lin-1 (Ets transcription factor)/Lin-31 inhibitory complex to specify vulval cell fate (35). In Drosophila, antibodies to the activated form of ERK detected dynamic expression patterns that correlated with known RTK pathways (13). In mouse, fetal submandibular gland branching morphogenesis was enhanced by EGF and inhibited by the MEK inhibitor PD-98059 (17). These studies suggest that ERK-1/2 signaling is tightly regulated during development and that this regulation is evolutionarily conserved. The observation that inhibition of ERK-1/2 signaling reduces branching morphogenesis in vitro suggests that pulmonary hypoplasia in vivo may involve downregulation of ERK-1/2 signaling. This hypothesis is supported by observations that ERK-1/2 activities are downregulated in the rat model of nitrofen-induced pulmonary hypoplasia associated with congenital diaphragmatic hernia (20). However, the mechanisms of ERK-1/2-mediated alterations in hypoplasia are not well understood and are currently being investigated. Signaling pathways involving ERK-1/2, ERK-5, and phosphatidylinositol 3-kinase (PI3 kinase) are important in regulating specific aspects of morphogenesis in other branching organs (16, 27). For example, renal inner medullary collecting duct (mIMCD-3) cells undergo in vitro branching tubulogenesis in response to both the c-met receptor ligand HGF and EGF receptor ligands. Activation of the ERK-1/2 signaling pathway is critical for HGF-induced cell motility/morphogenesis, whereas ERK-5 appears to be required for EGFdependent morphogenesis (16). Another example of pathway-specific morphogenic programming involves the EpH4 mammary epithelial cells (27). When cultured in Matrigel, they form branched tubules in the presence of HGF, but form alveolar structures in the presence of neuregulin, a ligand of c-erbB tyrosine kinase receptors. The HGF-induced branching required PI3 kinase signaling as it was abrogated by the PI3 kinase inhibitors wortmannin and LY-294002 and could be induced by transfection of a c-met-specific substrate, Gab1, which activated the PI3 kinase pathway. In contrast, neuregulin-induced alveolar morphogenesis required MEK/ERK signaling because it was inhibited by the MAP kinase inhibitor PD-98059 and activated by a hybrid receptor containing the intracellular domain of the c-erbB2 receptor. Thus c-met and c-erbB2 elicit distinct morphogenic programs in mammary epithelial cells; formation of branched tubules relies on a pathway involving PI3 kinase, whereas 282 • MARCH 2002 •

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Fig. 3. Inhibition of MAP kinases with U-0126 induces apoptosis in mesenchymal tissue using terminal deoxynucleotidyltransferase dUTP nick end labeling (TUNEL) assay. Cross sections of rat embryonic day 13 (E13) lung explants grown in the presence of DMSO (negative control; A) or U-0126 (B) for 4 days. Sections were labeled using a TUNEL (green fluorescence) assay and the nuclear stain propidium iodide (PI; red). C: negative controls for TUNEL assay, absent the deoxynucleotidyltransferase enzyme. Effects of the caspase inhibitor z-Val-AlaAsp(OCH3)-fluoromethylketone (zVAD-FMK) on background apoptosis (D) and U-0126-induced apoptosis (E) are shown. Magnification, ⫻125. The last 2 images in rows A and B are digitally enlarged (1.7-fold) regions from the adjacent photomicrographs to highlight mesenchymal apoptosis in the U-0126-treated tissue; mesenchyme (m) and epithelium (e) are indicated.

alveolar morphogenesis requires MEK. Thus individual signaling networks control program-specific morphogenesis. Our observation that inhibition of MEK/ERK signaling with U-0126 leads to caspase-dependent apoptosis suggeststhatdecreasedERK-1/2signalinginducesapoptosis and is consistent with other studies (2, 22, 31). In HeLa cells, inhibition of ERK signaling with the MEK inhibitor PD-98059 led to caspase-dependent apoptosis and induced p38 activity, while JNK activities remained unchanged, as in the present study (2). Inhibition of ERK-1/2 with U-0126 blocked proliferation of neural progenitor cells while increasing the population of cells undergoing apoptosis (22). In addition, a U-0126mediated dose-dependent activation of caspase-3, cleavage of 116-kDa poly(ADP-ribose) polymerase, and morphological signs of apoptosis were observed in human chondrocytes (31), suggesting that MEK/ERK inhibiAJP-Lung Cell Mol Physiol • VOL

tion can lead to increased apoptosis in a broad range of cell types. Although our results indicate that increased mesenchymal cell apoptosis is correlated with a reduction in fetal lung branching, evidence suggests that at least some apoptosis is necessary for proper growth and development (4, 23, 36). For example, dihydrotestosterone stimulates apoptosis of the mesenchyme surrounding the lung epithelial branch points and increases branching morphogenesis in mouse fetal lung explants (23). Conversely, the phosphatase inhibitor okadaic acid reduces apoptosis in a dose-dependent manner in the mesenchyme and in the epithelium as measured by TUNEL and inhibits branching morphogenesis in fetal rat lung cultures (36). Although these studies were conducted in fetal lung explant cultures, levels of apoptosis in vitro were similar to those observed in fetal lungs in vivo 282 • MARCH 2002 •

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Fig. 4. Caspase-3 activation by U-0126 treatment. Immunofluorescence analysis of activated caspase-3, a marker for apoptosis. Fetal lung explants were treated with DMSO (A) or U-0126 (B), and active caspase-3 was detected with a specific antibody. C: experimental control (no primary antibody). Hoechst, Hoechst-33342 trihydrochloride trihydrate, a nuclear stain.

(4). Thus it appears that apoptosis is perhaps a requirement for lung branching morphogenesis, and too much or too little disrupts a delicate balance and can lead to abnormal development. At least two factors contribute to the hypoplastic phenotype observed in U-0126-treated explants: reduced proliferation of the epithelium and increased apoptosis in the mesenchyme. These results suggest that ERK-1/2 signaling controls unique cell-fate pathways in different cell populations. Alternatively, it is possible that U-0126 affects only one cell type

and that other cell populations are affected via paracrine interactions. However, the intercellular apoptotic signal FasL does not appear to be involved in the induction of mesenchymal cell programmed cell death in our system (unpublished observations), which is consistent with its role in epithelium-specific apoptosis in developing lung (6). If it is necessary to orchestrate mesenchymal apoptosis and epithelial proliferation for proper branching, the role of the ERK-1/2 signaling provides a handle for deeper dissection of this coordination and may aid in the

Fig. 5. Proliferating cell nuclear antigen (PCNA) expression in methanolfixed explants. Cross sections of explants probed with PCNA monoclonal antibodies show cells undergoing normal mitosis when treated with DMSO (control; A), but reduced mitosis when treated with U-0126 (B). C: experimental controls (no PCNA antibody). Arrows indicate epithelium (e) and mesenchyme (m).

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discovery of possible therapeutic solutions to hypoplastic lung abnormalities. We thank T. Manganaro for technical help and Dr. A. Alessandrini for insightful experimental advice. This work was supported by National Heart, Lung, and Blood Institute Grant HL-62615 to J. J. Schnitzer. Partial funding was provided by National Institute of Child Health and Human Development Grant P01 HD-39942 to J. J. Schnitzer, T. B. Kinane, and P. K. Donahoe.

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