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Am J Physiol Lung Cell Mol Physiol 291: L820-L827, 2006. First published June 2, 2006; doi:10.1152/ajplung.00068.2006
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Strain-induced fetal type II epithelial cell differentiation is mediated via cAMP-PKA-dependent signaling pathway

Yulian Wang,1,* Benjamin S. Maciejewski,1,* Nicole Lee,1 Ophira Silbert,1 Nathan L. McKnight,2 John A. Frangos,2 and Juan Sanchez-Esteban1

1Department of Pediatrics, Division of Neonatology, Women and Infants Hospital of Rhode Island, Brown Medical School, Providence, Rhode Island; 2La Jolla Bioengineering Institute, La Jolla, California

Submitted 22 February 2006 ; accepted in final form 26 May 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The signaling pathways by which mechanical forces modulate fetal lung development remain largely unknown. In the present study, we tested the hypothesis that strain-induced fetal type II cell differentiation is mediated via the cAMP signaling pathway. Freshly isolated E19 fetal type II epithelial cells were cultured on collagen-coated silastic membranes and exposed to mechanical strain for varying intervals, to simulate mechanical forces during lung development. Unstretched samples were used as controls. Mechanical strain activated heterotrimeric G-protein {alpha}s subunit, cAMP, and the transcription factor cAMP response element binding protein (CREB). Incubation of E19 cells with the PKA inhibitor H-89 significantly decreased strain-induced CREB phosphorylation. Moreover, adenylate cyclase 5 and CREB genes were also mechanically induced. In contrast, components of the PKA-independent (Epac) pathway, including Rap-1 or B-Raf, were not phosphorylated by strain. The addition of forskolin or dibutyryl cAMP to unstretched E19 monolayers markedly upregulated expression of the type II cell differentiation marker surfactant protein C, whereas the Epac agonist 8-pCPT-2'-O-Me-cAMP had no effect. Furthermore, incubation of E19 cells with the PKA inhibitor Rp-2'-O-monobutyryladenosine 3',5'-cyclic monophosphorothioate or transient transfection with plasmid DNA containing a PKA inhibitor expression vector significantly decreased strain-induced surfactant protein C mRNA expression. In conclusion, these studies indicate that the cAMP-PKA-dependent signaling pathway is activated by force in fetal type II cells and participates in strain-induced fetal type II cell differentiation.

stretch; surfactant protein C; mechanotransduction

THE DEVELOPMENT OF NORMAL pulmonary alveolar epithelium during fetal life is critical for successful adaptation to extrauterine life. This key process in lung development requires the differentiation of the distal epithelium into type II epithelial cells, which produce surfactant and prevent alveolar collapse during expiration. Even though it is well documented that fetal lung development is regulated by mechanical forces generated in utero by repetitive breathing movements and by fluid distension (12, 22, 28, 40–42, 48, 51), the molecular and cellular mechanisms by which mechanical forces promote lung development remain largely unknown. Past studies have begun to identify mechanoreceptors and mechanically trigged signaling pathways involved in lung growth and differentiation. For example, strain-induced fetal type II epithelial cell proliferation is mediated via platelet-derived growth factor receptor B, calcium channels, and protein kinase C [see reviews (28, 29, 51)]. On the other hand, integrins (43), epidermal growth factor receptor, and the ERK pathway have been shown to participate in strain-induced fetal type II cell maturation (28, 40–44). Nevertheless, further investigations are still needed to unveil the complexity of mechanotransduction during fetal lung development.

The second messenger cAMP regulates important cellular functions, including proliferation, differentiation, and apoptosis (47). cAMP is generated from intracellular ATP by adenylyl cyclases after heterotrimeric G{alpha}s protein activation. Several lines of evidence indicate that cAMP signaling participates in alveolar fluid reabsorption (26), airway smooth muscle relaxation (21), endothelial barrier permeability (34) and surfactant secretion (38). Importantly, cAMP agonists stimulate surfactant phospholipid synthesis (16) and induce alveolar type II cell differentiation in cultured fetal lung explants (2, 14). Moreover, cAMP is required for maintenance of human fetal type II cell phenotype in vitro (1, 13). Together, these studies suggest that cAMP may be an important mediator of lung development in vivo.

cAMP-dependent protein kinase (PKA), a key cellular target for cAMP, is composed of two regulatory and two catalytic subunits. Binding of cAMP to the regulatory subunits leads to a conformational change and dissociation of the catalytic subunits, which become active and phosphorylate serine and/or threonine residues on specific substrate proteins (47). The cAMP response element binding protein (CREB) transcription factor is a major nuclear target for the catalytic subunit of PKA (31). Although PKA is generally recognized as being the primary effector of cAMP signaling, several other cAMP-binding proteins have now been described, including the cyclic nucleotide-gated channels involved in transduction of olfactory and visual signals (15, 24), and cAMP-activated guanine-nucleotide exchange factor Epac, which activates the monomeric G protein Rap-1 (9). Thus, depending on the nature and organization of cAMP-binding proteins expressed in different cell types, the effects of cAMP may be mediated by PKA-dependent or -independent pathways (25).

Therefore, based on the role of cAMP in lung development, the objective of this study was to assess whether cAMP participates in mechanotransduction of fetal type II cells and to identify downstream effector proteins. In addition, we analyzed whether these signaling pathways mediate fetal type II cell differentiation. We used an in vitro model system in which cultured E19 type II cells are exposed to a physiologically relevant level of mechanical strain similar to that observed during lung development.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell isolation and mechanical distension protocol. Fetal rat lungs were obtained from timed-pregnant Sprague-Dawley rats (Charles Rivers, Wilmington, MA), and E19 type II cells were isolated as previously described (43). Animal experiments were performed in compliance with Institutional Animal Care and Use Committee guidelines. Epithelial cells were then seeded onto Bioflex silicone elastomer multiwell plates precoated with collagen 1 (Flexcell, Hillsborough, NC) and maintained in serum-free DMEM for 24 h. Plates containing adherent cells were mounted in a Flexercell FX-4000 strain unit (Flexcell). For Northern blot or real-time PCR (qRT-PCR) experiments, equibiaxial elongation of 5% was applied at intervals of 60 cycles/min for 15 min plus 2.5% continuous distension for the remaining 45 min of each hour for 16 h. This regimen was chosen to simulate mechanical forces experienced by type II epithelial cells during fetal lung development (37). Cells grown on nonstrained substrates were treated in an identical manner and served as controls.

Heterotrimeric G protein {alpha}s-subunit activation assay. The 32P-labeled, nonhydrolyzable, photoreactive GTP analog azidoanilide-GTP (AAGTP; specific activity of 25–50 Ci/mmol, Affinity Labeling Technologies) was utilized to detect G protein {alpha}s-subunit protein activation (19). Subconfluent E19 type II cell monolayers seeded on collagen-coated bioflex plates were incubated in buffer (10 mM HEPES, pH 7.2, 135 mM KCl, 10 mM MgCl2, 5 mM EGTA, 5 mM glucose, and 1 mg/ml BSA) containing digitonin (20 µM; Sigma) for 3 min at 37°C. Digitonin is a detergent that permeabilizes the cell membrane and facilitates the incorporation of AAGTP into the cells. Monolayers were then incubated in 100 µl/well of buffer containing guanosine 5'-(beta-thio)diphosphate (Calbiochem) and 10–15 µCi AAGTP (1.64 nM/106 cells) at 1:1 to 1:10 (mol/mol) with AAGTP for 1 min at 37°C. Cells were then exposed to 5% cyclic strain (60 cycles for 1 min). Nonstrained monolayers were otherwise treated in an identical manner and served as controls. After the appropriate stretch or control protocol, plates were placed on ice and cells were immediately irradiated for 1 min with ultraviolet light (UVP, model UVG-54; 254-nm wavelength) at ~3 cm above the membranes to covalently bind the radiolabeled GTP. Monolayers were then rinsed with ice-cold PBS with 4 mM dithiothreitol (Sigma) to reduce nonspecific cross-linking of the photolabel. For the identification of the AAGTP-labeled G protein {alpha}s-subunit (G{alpha}s) by immunoprecipitation, cells were immediately lysed in RIPA buffer with protease inhibitors as described below. Lysates were centrifuged, and equal amounts of proteins were incubated with agarose bead-conjugated anti-G{alpha}s polyclonal antibody (Sigma) overnight at 4°C and washed four times with RIPA buffer. Immunoprecipitates were solubilized in electrophoresis sample buffer and analyzed by SDS-PAGE with 4% acrylamide stacking and 10% acrylamide separating gel. After electrophoresis, gels were dried and then exposed to Kodak XR-OMAT film with an intensifying screen for 1–2 days at –80°C. Autoradiographs were quantified by densitometry.

cAMP ELISA assay. A nonacetylation assay kit (cAMP Biotrak enzyme immunoasay system, no. RPN225; Amersham Biosciences) was used to determine whether mechanical strain increases intracellular levels of cAMP. This assay is based on competition between unlabeled cAMP and a fixed quantity of peroxidase-labeled cAMP for a limited number of binding sites on a cAMP-specific antibody. Briefly, E19 type II cells cultured on collagen 1 substrates were preincubated with IBMX (1 mM) and then exposed to the mechanical strain regimen for different periods of time. Monolayers were lysed in lysis reagent 1 (3.5% of dodecyltrimethylammonium bromide in assay buffer containing 0.05 M sodium acetate, pH = 5.8 and 0.02% BSA) for 10 min. Standards and samples were loaded into 96-well plates coated with donkey anti-rabbit IgG. Plates were incubated with rabbit anti-cAMP antibody for 2 h at 2°C. cAMP-peroxidase conjugate was then added for 1 h at the same temperature. Wells were washed four times and incubated with tetramethylbenzidene substrate for 30 min at room temperature. Reactions were stopped by adding 1 M sulfuric acid. Optical density was determined in a plate reader at 450 nm. Values (fmol/µg of protein) were derived from a standard curve.

Rap-1 activation assay. Rap-1 activation was assayed by affinity precipitation using a kit from Upstate (Lake Placid, NY) according to the manufacturer's recommendations. Briefly, E19 cells were exposed to 5% cyclic strain for up to 15 min. Cells were lysed with TBL buffer (50 mM Tris·HCl, pH 7.4, 0.5 M NaCl, 1% NP-40, 2.5 mM MgCl2, 10% glycerol); extracted proteins were incubated for 45 min at 4°C with 20 µg of Ral GDS-rap binding domain (RBD) agarose slurry. Agarose beads were precipitated, washed, and analyzed for bound GTP-Rap1 by 12% SDS-PAGE and Western blot with anti-Rap-1 antibody.

B-Raf kinase assay. B-Raf phosphorylation was analyzed by an in vitro kinase assay kit using MEK1 as a substrate (Upstate). Samples were incubated for 30 min at 30°C in a kinase reaction containing magnesium-ATP cocktail, active B-Raf, and unactive MEK1. The reaction was stopped by addition of 5x SDS Laemmli sample buffer and resolved on SDS-PAGE. B-Raf activity was analyzed by Western blot using anti-phospho-MEK1/2 antibody.

Assessment of CREB phosphorylation. To measure CREB activity, cell monolayers were lysed with ice-cold RIPA buffer (150 mM NaCl, 100 mM Tris-base, pH 7.5, 1% deoxycholate, 0.1% SDS, 1% Triton X-100, 3.5 mM Na3VO4, 2 mM PMSF, 50 mM NaF, and 100 mM sodium pyrophosphate) with protease inhibitors (10 µg/ml leupeptin, 10 µg/ml aprotinin, and 143.5 µM aminoethyl benzenesulfonyl fluoride). Lysates were centrifuged, and total protein contents were determined by the bicinchoninic acid method. Protein samples were separated by one-dimensional SDS-PAGE and transferred to polyvinylidene fluoride membranes (Bio-Rad). Membranes were incubated for 1 h at room temperature in blocking buffer (Tris-buffered saline-Tween 20 with 5% nonfat dry milk) and then incubated with anti-phospho-CREB (serine residue 133) monoclonal antibody (Cell Signaling) for 1 h at room temperature. After a washing period, secondary antibody (donkey anti-rabbit-horseradish peroxidase, diluted 1:2,000 in blocking buffer) was added for 1 h at room temperature. Immunoreactive phospho-CREB was detected by enhanced chemiluminescence (Amersham). To control for protein loading, membranes were stripped and reprobed with antibody to total CREB (Cell Signaling); the intensity of the bands was analyzed by densitometry.

Real-time PCR. Total RNA was isolated from E19 type II cells by a single-step method as previously described (41) and purified further with the Turbo DNA-free kit (Ambion). RNA (3 µg) was reverse transcribed into cDNA by the Script cDNA synthesis kit (Bio-Rad) according to the manufacturer's instructions. Predesigned TaqMan primers were purchased from Assays-on-Demand gene expression products (Applied Biosystems). The following primers were used: beta2-microglobulin (Rn00560865_m1), 18S (Hs99999901_s1), adenylate cyclase 5 (Rn00575059_m1), CREB binding protein (Rn00591291_m1), and surfactant protein (SP)-C (Rn00569225_m1). To amplify the cDNA by qRT-PCR, 5 µl of the resulting cDNA were added to a mixture of 25 µl of TaqMan Universal PCR Master Mix (Applied Biosystems) and 2.5 µl of 20x Assays-on-Demand gene expression assay mix containing forward and reverse primers and TaqMan-labeled probe (Applied Biosystems). Standard curves were generated for each primer set and housekeeping genes beta2-microglobulin and 18S. Linear regression revealed efficiencies between 96 and 99%. The reactions were performed in an ABI Prism 7000 sequence detection system (Applied Biosystems) with an initial denaturation for 2 min at 50°C and 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. All assays were performed in triplicate.

Northern blot analysis. SP-C mRNA expression was detected by Northern blot as previously described in detail (41).

Transient transfection by electroporation. Fetal type II cells were transiently transfected by Amaxa's nucleofector technology (Amaxa Biosystems). Freshly isolated E19 type II epithelial cells were plated overnight on T75 flasks. Cells were harvested the next day by trypsinization, and aliquots of 2 x 106 cells in RPMI 1640 with 10% FBS were centrifugated at 100 g for 10 min. Supernatants were discarded, and cell pellets were resuspended in 100 µl of basic nucleofector solution (primary mammalian epithelial cell protocol; Amaxa Biosystems). Samples were mixed with 2 µg of plasmid DNA containing a PKA inhibitor (PKI) expression vector (a generous gift from Dr. Richard A. Maurer, University of Oregon), transferred into the appropriate cuvettes, and subjected to electrical pulses with the use of the Amaxa Nucleofactor II apparatus (Amaxa Biosystems). Samples containing no DNA were otherwise treated in an identical manner and served as negative controls (pulse only). On the basis of preliminary experiments, a T-13 program from the Nucleojector device was selected. After electroporation, samples were immediately transferred into Eppendorf tubes containing prewarmed RPMI 1640 plus 10% FBS and incubated at 37°C for 10 min. Cells were then transferred into bioflex plates precoated with collagen and left undisturbed for 24 h in a culture incubator. Cell viability 24 h after electroporation was ~35%. Transfection efficiency using the control vector pmaxGFP was ~50%. Twenty-four hours after transfection, monolayers were exposed to mechanical strain for 16 h.

Statistical analysis. Results are expressed as means ± SE from at least three experiments, using different rat litters for each experiment. Control and strained samples were compared by unpaired Student's t-test. For multiple comparisons, data were analyzed with ANOVA followed by post hoc tests, and Instat 3.0 (GraphPad Software, San Diego, CA) was used for statistical analysis. P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Mechanical strain activates G{alpha}s-subunit in fetal type II cells. Because of the importance of heterotrimeric G proteins for mechanotransduction shown in other cell types (4, 19), we examined whether G proteins, specifically the G{alpha}s-subunit, are activated in fetal type II epithelial cells. E19 type II cell monolayers cultured on silastic membranes coated with collagen 1 were incubated with the photoactive GTP analog AAGTP. To enhance the signal-to-noise ratio, the nonhydrolyzable GDP analog guanosine 5'-(beta-thio)diphosphate, which irreversibly binds to GTP-binding proteins, was incubated with AAGTP at different concentrations. Subconfluent E19 cells were then exposed to 5% cyclic strain for 1 min. Our results showed that mechanical strain activates the G{alpha}s-subunit by twofold at a GDP-to-AAGTP ratio of 1:1 (Fig. 1). The addition of nonradiolabeled GTP at 100 µM during AAGTP incubation competitively blocked strain-induced G{alpha}s activation, demonstrating the specificity of the antibody (data not shown).


Figure 1
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  		Fig. 1. Mechanical strain activates G protein {alpha}s-subunit. E19 type II cells were incubated with azidoanilide-GTP (AAGTP) and GDP at different ratios and exposed to cyclic strain for 1 min. After ultraviolet irradiation to covalently bind AAGTP, proteins were immunoprecipitated with anti-G{alpha}s antibody and analyzed by SDS-PAGE. Gels were dried and exposed to film with an intensifying screen. Top: representative Western blot and Coomassie blue gel staining to control for protein loading. Bottom: results are means ± SE from 3 different experiments at guanosine 5'-(beta-thio)diphosphate-to-AAGTP ratio of 1:1 (*P < 0.01).

 
Mechanical strain increases intracellular cAMP levels. Because the heterotrimeric G{alpha}s-subunit regulates the activity of the second-messenger cAMP, we next studied whether cAMP is stimulated by mechanical strain in fetal type II cells. E19 monolayers were preincubated with the phosphodiesterase inhibitor IBMX and then exposed to mechanical strain for different lengths of time; cAMP activation was analyzed by ELISA. These studies revealed that intracellular cAMP activation was initially detected after 15 min of strain, reached its highest level by 30 min (by threefold), and returned to the baseline by 2 h (Fig. 2). cAMP increased by 5 min of strain in monolayers not incubated with IBMX (data not shown).


Figure 2
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  		Fig. 2. Mechanical strain stimulates cAMP. E19 type II cell monolayers cultured on collagen 1 substrates were preincubated with IBMX (1 mM) and then exposed to mechanical strain, as described in MATERIALS AND METHODS, for the indicated periods of time. Intracellular cAMP levels were determined by ELISA. Results are means ± SE from 3 different experiments (*P < 0.05; **P < 0.01).

 
Mechanical strain activates the PKA-dependent signaling pathway in fetal type II epithelial cells. Next, we studied cAMP-binding proteins activated by force. A guanine-nucleotide exchange factor for Rap-1 (Epac) has been identified to be activated by cAMP in a PKA-independent manner (9). Rap-1 activates the ERK pathway via B-Raf. Because of the important role of ERK in strain-induced type II cell differentiation (44), we examined whether this cascade is activated via Epac. Our results revealed that cyclic mechanical strain activates neither Rap-1 nor B-Raf in fetal type II epithelial cells (Fig. 3). In contrast, mechanical strain maximally phosphorylated the transcription factor CREB, a major nuclear target for the catalytic subunit of the PKA, by twofold after 30 min of mechanical strain (Fig. 4A). Preincubation of E19 monolayers with the PKA inhibitor H-89 significantly decreased strain-induced CREB stimulation (Fig. 4B), indicating that activation of CREB by force is, at least in part, mediated via PKA. In addition, CREB (control = 51.37 ± 2.31, strain = 77.89 ± 5.44; P = 0.01) and adenylate cyclase 5 genes (control = 1.44 ± 0.22, strain = 3.66 ± 0.16; P = 0.001) were upregulated by 50% and 2.5-fold, respectively, after 16 h of mechanical strain, as measured by qRT-PCR as measured by qRT-PCR, relative quantification (Fig. 5). Together, these studies suggest that mechanical strain activates specifically the PKA-dependent pathway, whereas the Epac cascade is not stimulated by force in fetal type II epithelial cells.


Figure 3
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  		Fig. 3. Mechanical strain phosphorylates neither Rap-1 nor B-Raf. Subconfluent E19 type II cells were exposed to 5% cyclic strain for up to 15 min. Extracted proteins were processed to detect Rap-1 and B-Raf activation by affinity precipitation assay and by an in vitro kinase assay, respectively. Blots are representative of 2 separate experiments.

 

Figure 4
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  		Fig. 4. A, top: Western blot showing that mechanical strain phosphorylates cAMP response element binding protein (CREB) in fetal type II pulmonary epithelial cells. Type II cells isolated from E19 rat fetal lungs were cultured on flexible silastic membranes coated with collagen 1 and then subjected to cyclical mechanical strain for up to 30 min. Proteins were extracted, and the levels of CREB phosphorylation (p-CREB) were determined by using a phospho-specific CREB antibody. Blots were then stripped and incubated with total CREB (t-CREB) antibody to control for protein loading. A, bottom: results from 3 experiments showing that mechanical strain maximally phosphorylates CREB after 30 min of strain (*P < 0.01; **P < 0.05). B: effects of PKA inhibitor on CREB phosphorylation. E19 cells were preincubated with the PKA inhibitor H-89 (10 µM) and then exposed to cyclic strain for 30 min. CREB activation was assessed by Western blot. Data are representative of 3 separate experiments (*P < 0.01; **P < 0.05).

 

Figure 5
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  		Fig. 5. Representative qRT-PCR amplification plot comparing CREB (2) and adenylate cyclase 5 (3) mRNA expressions in E19 type II epithelial cell exposed to mechanical strain (S) for 16 h and unstrained control samples (C). beta2-Microblobulin (1) was used as a housekeeping gene (n = 3, using different rat litters for each experiment).

 
Upregulation of SP-C by cAMP agonists is not mediated via the Epac pathway. From previous studies demonstrating that cAMP agonists induce fetal type II epithelial cell differentiation in vitro (2, 14), we next studied whether this effect is mediated via a PKA-dependent or -independent pathway. Freshly isolated E19 cells cultured on collagen substrates were incubated with cAMP agonists forskolin or 8-pCPT-2'-O-Me-cAMP and maintained under nonstrained conditions for 16 h. SP-C mRNA abundance was analyzed by Northern blot. Figure 6 shows that the addition of forskolin (25 µM) significantly increased SP-C expression. In contrast, 8-pCPT-2'-O-Me-cAMP, a cAMP agonist that selectively activates Epac, at 50 µM (Fig. 6, lane 3) or 100 µM (Fig. 6, lane 4) had not effect. These data indicate that upregulation of SP-C by cAMP agonists is not mediated via Epac.


Figure 6
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  		Fig. 6. A representative Northern blot from 2 separate experiments showing that the addition of forskolin (25 µM) to nonstrained E19 type II cells for 16 h markedly increases surfactant protein (SP)-C mRNA expression, whereas incubation with 8-pCPT-2'-O-Me-cAMP (50 µM and 100 µM, lanes 3 and 4, respectively), a cAMP agonist specific for EPAC-B-Raf pathway, has no effects.

 
Time-course effects of cAMP agonists and strain on SP-C mRNA expression. To establish a closer chronological association between activation of the cAMP-PKA pathway and its effect on SP-C expression, we performed experiments to assess time-course induction of SP-C mediated by cAMP agonists or by force. E19 monolayers were incubated with dibutyryl cAMP (200 µM) or forskolin (25 µM) or exposed to mechanical strain for up to 8 h. SP-C mRNA expression was analyzed by Northern blot. Figure 7A shows that an increase of SP-C mRNA by cAMP agonists was first observed after 4–6 h of incubation. Likewise, upregulation of SP-C mRNA was detected as early as 4 h after mechanical strain (Fig. 7B) and maximally induced by 16 h (Fig. 8). Together, these data support a temporal relationship between stimulation of the cAMP-PKA cascade and upregulation of SP-C mRNA mediated by cAMP agonists or by force.


Figure 7
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  		Fig. 7. A: time-course effect of cAMP agonists on SP-C mRNA expression. E19 monolayers were incubated with dibutyryl cAMP (Bt2cAMP; 200 µM) or forskolin (25 µM) for the indicated periods of time. SP-C mRNA expression levels were determined by Northern blots. Results are from 3 separate experiments (*P < 0.05). B: time-course effect of mechanical strain on SP-C mRNA expression. E19 type II cells were subjected to mechanical strain for up to 8 h. SP-C mRNA was detected by Northern blot. Data from 3 separate experiments are normalized to 18S rRNA concentration (*P < 0.05).

 

Figure 8
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  		Fig. 8. A: effects of PKA inhibitor on strain-induced SP-C mRNA. A representative Northern blot (top) and statistical analysis (bottom) from 3 separate experiments show that the PKA inhibitor Rp-2'-O-monobutyryladenosine 3',5'-cyclic monophosphorothioate (Rp-8-MB-cAMPS; 200 µM) significantly decreases strain-induced SP-C mRNA expression (*P < 0.01; **P < 0.05). B: transient transfection with a PKA inhibitor expression vector inhibits strain-induced SP-C mRNA expression. E19 type II cells were transiently transfected by electroporation as described in MATERIALS AND METHODS and exposed to mechanical strain for 16 h. SP-C mRNA abundance was determined by qRT-PCR [pulse only (no plasmid DNA)]. Data from 3 experiments are normalized and expressed as relative quantification values (*P < 0.01).

 
Strain-induced SP-C mRNA expression is mediated via PKA. To determine whether the PKA-dependent signaling pathway participates in strain-induced type II cell differentiation, E19 cells were preincubated with the PKA inhibitor Rp-2'-O-monobutyryladenosine 3',5'-cyclic monophosphorothioate (Rp-8-MB-cAMPS) (200 µM), which inhibits dissociation of the catalytic subunit from the regulatory subunit or transiently transfected with plasmid DNA containing a PKI expression vector. E19 monolayers were then exposed to mechanical strain for 16 h. Figure 8 shows that strain-induced upregulation of SP-C is significantly inhibited in E19 cells treated with Rp-8-MB-cAMPS or transfected with PKI, respectively. These data suggest that strain-induced type II cell differentiation, as measured by SP-C mRNA expression, is mediated via PKA.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Although mechanical forces are essential for normal lung development, the cell signaling pathways mediating this process have not been fully established. In the present study, we analyzed the role of the second messenger cAMP and its binding-associated proteins in mechanotransduction of fetal type II epithelial cells. Our results showed that mechanical strain activates heterotrimeric G{alpha}s-subunit, cAMP, and the transcription factor CREB, whereas components of the PKA-independent pathway such as Rap-1 or B-Raf were not stimulated by force. In addition, strain-induced type II cell differentiation, using SP-C as a marker, was found to be mediated via PKA.

Heterotrimeric G proteins play a pivotal role in cell signaling by connecting cell surface receptors to a variety of intracellular signaling pathways (20). The role of G protein signaling in mechanotransduction has been previously described in other cell types (4, 5, 18, 19). Indirect evidence also suggests the involvement of G proteins in alveolar epithelial cells exposed to strain (7). In the present study, using the photolabile GTP analog, we directly demonstrated the rapid activation of G{alpha}s-subunit in response to mechanical strain. These data also support a key role for G proteins in mechanotransduction of fetal type II epithelial cells. Although a wide range of extracellular signals are known to stimulate G{alpha}s via G protein-coupled receptors (35), the receptors coupling mechanical signals to G proteins are for the most part unknown. Mechanical stresses applied to integrin receptors activate G proteins and the cAMP signaling cascade (32). However, past investigations showed the ability of the membrane phospholipid bilayer to mediate shear-stress activation of G proteins in the absence of receptors (17), suggesting that cell membrane deformation by mechanical forces may be sufficient to stimulate G proteins.

Increase of intracellular cAMP levels is triggered by G{alpha}s subunit stimulation.

The present investigation found that the second messenger cAMP is activated in fetal type II epithelial cells by strain. These findings corroborate previous experiments showing that cAMP levels increased in lung tissues after partial pneumonectomy (39), in organotypic cultures of fetal rat lung cells (46), and in fetal rabbit epithelial cells (45) exposed to mechanical strain. In contrast, other studies found the cAMP content was not affected by force (30). Our data support a role for the second messenger cAMP in mechanotransduction during fetal lung development.

PKA is generally recognized as the primary effector of cAMP. However, other signaling targets are also regulated by cAMP, including proteins from the ERK pathway. cAMP can inhibit ERK signaling and cell proliferation by competing with Ras for Raf-1 and A-Raf binding sites (52). On the other hand, cAMP can stimulate ERK via Epac, Rap-1, and B-Raf pathway (50). Because past studies from our laboratory showed that the ERK pathway is activated by force and participates in strain-induced fetal type II cell differentiation (44), we assessed whether phosphorylation of ERK is mediated via Epac. Our data indicate that the PKA-independent pathway does not activate ERK.

In addition, cAMP agonists have been reported to increase SP-B gene expression in human, rat, and rabbit explant cultures, but effects on SP-C have been more modest and inconsistent (36, 49). Therefore, we tested whether cAMP agonists increase SP-C mRNA expression in cultured type II cells under unstrained conditions and whether the Epac pathway mediates this effect. We observed a significant increase on SP-C expression induced by dibutyryl cAMP or forskolin, but this maturational effect is not mediated via Epac because the cAMP agonist specific for Epac did not increase SP-C expression.

PKA is an important cellular target for cAMP.

PKA activity increased in rat after pneumonectomy and in isolated perfused lung distended by continuous positive airway pressure (39). These studies show that mechanical forces are important triggers for PKA stimulation. We were unable to consistently demonstrate activation of PKA by strain or by cAMP agonists such as forskolin. This may be due to high levels of PKA phosphorylation observed under basal conditions in our experimental system or rapid PKA dephosphorylation. To overcome this technical difficulty, we used an alternative approach by looking at the effects of PKA inhibitor on CREB phosphorylation. Our findings showed that phosphorylation of CREB by strain is mediated via PKA, since incubation with H-89, which competitively binds to the catalytic subunit of PKA, significantly decreased strain-induced CREB phosphorylation. These experiments provide indirect evidence of PKA activation by mechanical forces in fetal type II epithelial cells. Pharmacological blockade of the catalytic subunit of PKA or transient transfection with a construct containing the PKI expression vector also demonstrated that PKA participates in strain-induced SP-C mRNA expression. The heat-stable inhibitor of PKA (PKI) is a small protein that binds to the catalytic subunit of the kinase and inhibits its activity by preventing nuclear translocation (11). PKI is present in small amounts in a variety of mammalian tissues, including the lungs (6). It has been shown that transfection with a PKI construct inhibits cAMP-dependent transcription of several genes (8). Together, these studies suggest that the cAMP-PKA-dependent signaling pathway may be important for mechanotransduction of fetal type II epithelial cells and support previous investigations demonstrating the role of this pathway in alveolar type II cell differentiation.

Induction of gene expression by the second messenger cAMP is generally mediated by binding of CREB to a conserved sequence present in the promoter of many cAMP-responsive genes (33). CREB is activated by phosphorylation at serine residue 133 in response to a number of signaling pathways, including cAMP, calcium, and stress and mitogenic stimuli (23). The kinetics of CREB phosphorylation may be of crucial importance for activation of CREB-dependent transcription (23). CREB activation in response to cAMP agonists usually peaks after 20–30 min, coinciding with the time required for PKA catalytic subunit levels to become maximal in the nucleus (31). Consistent with these observations, we found a similar peak of CREB stimulation by strain. However, the participation of other signaling pathways in CREB stimulation, such as calcium signaling and ERK, cannot be ruled out. Furthermore, our studies are insufficient to demonstrate whether stimulation of CREB mediates gene transcription, since phosphorylation of multiple kinases may be required for full activation of CREB (23).

Phosphorylation of CREB was previously observed in bovine aortic endothelial cells exposed to cyclic strain (10). In addition, shear stress in endothelial cells increases the expression of early response genes such as c-fos and c-jun, which contain consensus sites for CREB binding (27). These studies suggest that CREB may participate in strain-induced gene expression. Although the CREB family of transcription factors plays an important role in mediating the transcriptional regulation of the SP-B (3) and SP-A genes (53), the potential role of the transcription factor CREB in mechanotransduction of fetal type II cells is presently unknown.

In summary, these studies demonstrate that mechanical forces activate heterotrimeric G{alpha}s protein and the cAMP-PKA-dependent signaling pathway in cultured fetal type II epithelial cells. We have also identified the participation of this pathway in strain-induced fetal type II epithelial cell differentiation. These investigations provide additional new information on signaling pathways activated by mechanical forces during fetal lung development. Future studies will be required to identify how these different signaling pathways are integrated at the transcriptional level to promote lung maturation.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institute of Health grant RR-018728. J. Sanchez-Esteban is a Parker B. Francis Fellow in Pulmonary Research.


   ACKNOWLEDGMENTS
 
The authors thank Brenda Vecchio for manuscript preparation.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. Sanchez-Esteban, Dept. of Pediatrics, Women & Infants Hospital, 101 Dudley St., Providence, RI 02905 (e-mail: jsanchezesteban{at}wihri.org)

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.

* Y. Wang and B. S. Maciejewski contributed equally to this work. Back


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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