Proteomic analysis of a murine model of lung hypoplasia induced by oligohydramnios.

Severe oligohydramnios (OH) due to prolonged loss of amniotic fluid can cause pulmonary hypoplasia. Animal model of pulmonary hypoplasia induced by amniotic fluid drainage is partly attributed to changes in mechanical compression of the lung. Although numerous studies on OH-model have demonstrated changes in several individual proteins, however, the underlying mechanisms for interrupting normal lung development in response to a decrease of amniotic fluid volume are not fully understood. In this study, we used a proteomic approach to explore differences in the expression of a wide range of proteins after induction of OH in a mouse model of pulmonary hypoplasia to find out the signaling/molecular pathways involved in fetal lung development. Liquid chromatography-massspectromery/mass spectrometry analysis found 474 proteins that were differentially expressed in OH-induced hypoplastic lungs in comparison to untouched (UnT) control. Among these proteins, we confirmed the downregulation of AKT1, SP-D, and CD200, and provided proof-of-concept for the first time about the potential role that these proteins could play in fetal lung development.

Mechanical stretch regulates the expression of specific miRNA in extracellular vesicles released from lung epithelial cells.

The underlying mechanism of normal lung organogenesis is not well understood. An increasing number of studies are demonstrating that extracellular vesicles (EVs) play critical roles in organ development by delivering microRNAs (miRNA) to neighboring and distant cells. miRNAs are important for fetal lung growth; however, the role of miRNA-EVs (miRNAs packaged inside the EVs) during fetal lung development is unexplored. The aim of this study was to examine the expression of miRNA-EVs in MLE-12, a murine lung epithelial cell line subjected to mechanical stretch in vitro with the long-term goal to investigate their potential role in the fetal lung development. Both cyclic and continuous mechanical stretch regulate miRNA differentially in EVs released from MLE-12 and intracellularly, demonstrating that mechanical signals regulate the expression of miRNA-EVs in lung epithelial cells. These results provide a proof-of-concept for the potential role that miRNA-EVs could play in the development of fetal lung.

Oligohydramnios compromises lung cells size and interferes with epithelial-endothelial development.

BACKGROUND AND OBJECTIVE: Severe oligohydramnios can induce pulmonary hypoplasia. However, the mechanisms by which leaking of fluids cause lung hypoplasia are not well defined. The objective of this study was to characterize a mouse model of pulmonary hypoplasia induced by oligohydramnios. METHODS: Amniotic sacs were punctured on E14.5 of gestation. Untouched fetuses were used as control. Pregnancy was allowed to continue until E18.5 in which lung tissue was collected and evaluated for morphometry, proliferation, differentiation, apoptosis, and angiogenesis. RESULTS: Our results found that lung weight, lung to total body weight ratio, and lung water content were reduced in oligohydramnios when compared to controls. In contrast, oligohydramnios did not affect the DNA content. Morphometric studies confirmed that oligohydramnios fetuses had smaller air spaces than control. Interestingly, cells from oligohydramnios fetuses have smaller size and less regular shapes. Oligohydramnios decreased the differentiation of type I epithelial cells and compromised apoptosis and angiogenesis while proliferation was not affected. CONCLUSIONS: Although, the smaller size of the lung could be explained by a decreased of lung fluids, our data suggest that increased of external compression secondary to severe oligohydramnios can compromise cell size and interfere with epithelial and endothelial development. Type I epithelial cells could have an unrecognized key role in the differentiation of the distal lung mediated by mechanical signals. Pediatr Pulmonol. 2017;52:746-756. © 2017 Wiley Periodicals, Inc.

Mechanotransduction as an Adaptation to Gravity.

Gravity has played a critical role in the development of terrestrial life. A key event in evolution has been the development of mechanisms to sense and transduce gravitational force into biological signals. The objective of this manuscript is to review how living organisms on Earth use mechanotransduction as an adaptation to gravity. Certain cells have evolved specialized structures, such as otoliths in hair cells of the inner ear and statoliths in plants, to respond directly to the force of gravity. By conducting studies in the reduced gravity of spaceflight (microgravity) or simulating microgravity in the laboratory, we have gained insights into how gravity might have changed life on Earth. We review how microgravity affects prokaryotic and eukaryotic cells at the cellular and molecular levels. Genomic studies in yeast have identified changes in genes involved in budding, cell polarity, and cell separation regulated by Ras, PI3K, and TOR signaling pathways. Moreover, transcriptomic analysis of late pregnant rats have revealed that microgravity affects genes that regulate circadian clocks, activate mechanotransduction pathways, and induce changes in immune response, metabolism, and cells proliferation. Importantly, these studies identified genes that modify chromatin structure and methylation, suggesting that long-term adaptation to gravity may be mediated by epigenetic modifications. Given that gravity represents a modification in mechanical stresses encounter by the cells, the tensegrity model of cytoskeletal architecture provides an excellent paradigm to explain how changes in the balance of forces, which are transmitted across transmembrane receptors and cytoskeleton, can influence intracellular signaling pathways and gene expression.

Mechanotransduction via TRPV4 regulates inflammation and differentiation in fetal mouse distal lung epithelial cells.

BACKGROUND: Mechanical ventilation plays a central role in the injury of premature lungs. However, the mechanisms by which mechanical signals trigger an inflammatory cascade to promote lung injury are not well-characterized. Transient receptor potential vanilloid 4 (TRPV4), a calcium-permeable mechanoreceptor channel has been shown to be a major determinant of ventilator-induced acute lung injury in adult models. However, the role of these channels as modulators of inflammation in immature lungs is unknown. In this study, we tested the hypothesis that TRPV4 channels are important mechanotransducers in fetal lung injury. METHODS: Expression of TRPV4 in the mouse fetal lung was investigated by immunohistochemistry, Western blot and qRT-PCR. Isolated fetal epithelial cells were exposed to mechanical stimulation using the Flexcell Strain Unit and inflammation and differentiation were analyzed by ELISA and SP-C mRNA, respectively. RESULTS: TRPV4 is developmentally regulated in the fetal mouse lung; it is expressed in the lung epithelium and increases with advanced gestation. In contrast, in isolated epithelial cells, TRPV4 expression is maximal at E17-E18 of gestation. Mechanical stretch increases TRPV4 in isolated fetal epithelial cells only during the canalicular stage of lung development. Using the TRPV4 agonist GSK1016790A, the antagonist HC-067047, and the cytokine IL-6 as a marker of inflammation, we observed that TRPV4 regulates release of IL-6 via p38 and ERK pathways. Interestingly, stretch-induced differentiation of fetal epithelial cells was also modulated by TRPV4. CONCLUSION: These studies demonstrate that TRPV4 may play an important role in the transduction of mechanical signals in the fetal lung epithelium by modulating not only inflammation but also the differentiation of fetal epithelial cells.

Epidermal growth factor receptor (EGFR) contributes to fetal lung fibroblast injury induced by mechanical stretch.

CONTEXT: Epidermal growth factor receptor (EGFR) is critical for normal fetal lung development. However, the role of this receptor in lung injury induced by mechanical ventilation is controversial. OBJECTIVE: To investigate in vitro whether EGFR plays a protective role or contributes to stretch-induced lung injury. METHODS: Fetal lung fibroblasts were isolated from wild-type and EGFR knockout mice and exposed to physiologic stretch (2.5% elongation) or injurious stretch (20% distention). Cells were evaluated for necrosis, apoptosis, proliferation and inflammation. RESULTS: Injurious stretch increased lactate dehydrogenase (LDH) release to similar degree in wild-type and knockout cells. In contrast, 20% stretch increased cleaved caspase-3 and decreased proliferating cell nuclear antigen (PCNA) only in wild-type cells. Furthermore, 20% stretch increased macrophage inflammatory protein-2 (MIP-2) and monocyte chemotactic protein-1 (MCP-1) by 3-5 fold in wild-type cells. In contrast, in knockout cells MIP-2 decreased by 50% and MCP-1 only increased by 60% when compared to physiologic stretch. CONCLUSION: Our data show a decrease of apoptosis and inflammation and absence of decreased proliferation after injurious stretch of fetal fibroblasts lacking EGFR. These data suggest that EGFR contributes to lung injury mediated by stretch. We speculate that EGFR may contribute to the arrest of lung development observed after mechanical ventilation by decreasing the population of lung fibroblasts.

Strain-induced differentiation of fetal type II epithelial cells is mediated via the integrin α6ß1-ADAM17/tumor necrosis factor-α-converting enzyme (TACE) signaling pathway.

Mechanical forces are critical for normal fetal lung development. However, the mechanisms regulating this process are not well-characterized. We hypothesized that strain-induced release of HB-EGF and TGF-α is mediated via integrin-ADAM17/TACE interactions. Employing an in vitro system to simulate mechanical forces in fetal lung development, we showed that mechanical strain of fetal epithelial cells actives TACE, releases HB-EGF and TGF-α, and promotes differentiation. In contrast, in samples incubated with the TACE inhibitor IC-3 or in cells isolated from TACE knock-out mice, mechanical strain did not release ligands or promote cell differentiation, which were both rescued after transfection of ADAM17. Cell adhesion assay and co-immunoprecipitation experiments in wild-type and TACE knock-out cells using several TACE constructs demonstrated not only that integrins α6 and ß1 bind to TACE via the disintegrin domain but also that mechanical strain enhances these interactions. Furthermore, force applied to these integrin receptors by magnetic beads activated TACE and shed HB-EGF and TGF-α. The contribution of integrins α6 and ß1 to differentiation of fetal epithelial cells by strain was demonstrated by blocking their binding site with specific antibodies and by culturing the cells on membranes coated with anti-integrin α6 and ß1 antibodies. In conclusion, mechanical strain releases HB-EGF and TGF-α and promotes fetal type II cell differentiation via α6ß1 integrin-ADAM17/TACE signaling pathway. These investigations provide novel mechanistic information on how mechanical forces promote fetal lung development and specifically differentiation of epithelial cells. This information could be also relevant to other tissues exposed to mechanical forces.

Reduced IL-10 production in fetal type II epithelial cells exposed to mechanical stretch is mediated via activation of IL-6-SOCS3 signaling pathway.

An imbalance between pro-inflammatory and anti-inflammatory cytokines is a key factor in the lung injury of premature infants exposed to mechanical ventilation. Previous studies have shown that lung cells exposed to stretch produces reduced amounts of the anti-inflammatory cytokine IL-10. The objective of these studies was to analyze the signaling mechanisms responsible for the decreased IL-10 production in fetal type II cells exposed to mechanical stretch. Fetal mouse type II epithelial cells isolated at embryonic day 18 were exposed to 20% stretch to simulate lung injury. We show that IL-10 receptor gene expression increased with gestational age. Mechanical stretch decreased not only IL-10 receptor gene expression but also IL-10 secretion. In contrast, mechanical stretch increased release of IL-6. We then investigated IL-10 signaling pathway-associated proteins and found that in wild-type cells, mechanical stretch decreased activation of JAK1 and TYK2 and increased STAT3 and SOCS3 activation. However, opposite effects were found in cells isolated from IL-10 knockout mice. Reduction in IL-6 secretion by stretch was observed in cells isolated from IL-10 null mice. To support the idea that stretch-induced SOCS3 expression via IL-6 leads to reduced IL-10 expression, siRNA-mediated inhibition of SOCS3 restored IL-10 secretion in cells exposed to stretch and decreased IL-6 secretion. Taken together, these studies suggest that the inhibitory effect of mechanical stretch on IL-10 secretion is mediated via activation of IL-6-STAT3-SOCS3 signaling pathway. SOCS3 could be a therapeutic target to increase IL-10 production in lung cells exposed to mechanical injury.

Stretch-induced fetal type II cell differentiation is mediated via ErbB1-ErbB4 interactions.

Stretch-induced differentiation of lung fetal type II epithelial cells is mediated through EGFR (ErbB1) via release of HB-EGF and TGF-α ligands. Employing an EGFR knock-out mice model, we further investigated the role of the ErbB family of receptors in mechanotranduction during lung development. Deletion of EGFR prevented endogenous and mechanical stretch-induced type II cell differentiation via the ERK pathway, which was rescued by overexpression of a constitutively active MEK. Interestingly, the expression of ErbB4, the only ErbB receptor that EGFR co-precipitates in wild-type cells, was decreased in EGFR-deficient type II cells. Similar to EGFR, ErbB4 was activated by stretch and participated in ERK phosphorylation and type II cell differentiation. However, neuregulin (NRG) or stretch-induced ErbB4 activation were blunted in EGFR-deficient cells and not rescued after ErbB4 overexpression, suggesting that induction of ErbB4 phosphorylation is EGFR-dependent. Finally, we addressed how shedding of ligands is regulated by EGFR. In knock-out cells, TGF-α, a ligand for EGFR, was not released by stretch, while HB-EGF, a ligand for EGFR and ErbB4, was shed by stretch although to a lower magnitude than in normal cells. Release of these ligands was inhibited by blocking EGFR and ERK pathway. In conclusion, our studies show that EGFR and ErbB4 regulate stretch-induced type II cell differentiation via ERK pathway. Interactions between these two receptors are important for mechanical signals in lung fetal type II cells. These studies provide novel insights into the cell signaling mechanisms regulating ErbB family receptors in lung cell differentiation.

An experimental system to study mechanotransduction in fetal lung cells.

Mechanical forces generated in utero by repetitive breathing-like movements and by fluid distension are critical for normal lung development. A key component of lung development is the differentiation of alveolar type II epithelial cells, the major source of pulmonary surfactant. These cells also participate in fluid homeostasis in the alveolar lumen, host defense, and injury repair. In addition, distal lung parenchyma cells can be directly exposed to exaggerated stretch during mechanical ventilation after birth. However, the precise molecular and cellular mechanisms by which lung cells sense mechanical stimuli to influence lung development and to promote lung injury are not completely understood. Here, we provide a simple and high purity method to isolate type II cells and fibroblasts from rodent fetal lungs. Then, we describe an in vitro system, The Flexcell Strain Unit, to provide mechanical stimulation to fetal cells, simulating mechanical forces in fetal lung development or lung injury. This experimental system provides an excellent tool to investigate molecular and cellular mechanisms in fetal lung cells exposed to stretch. Using this approach, our laboratory has identified several receptors and signaling proteins that participate in mechanotransduction in fetal lung development and lung injury.

Cigarette smoke causes lung vascular barrier dysfunction via oxidative stress-mediated inhibition of RhoA and focal adhesion kinase.

Cigarette smoke (CS) is a major cause of chronic lung and cardiovascular diseases. Recent studies indicate that tobacco use is also a risk factor for acute lung injury (ALI) associated with blunt trauma. Increased endothelial cell (EC) permeability is a hallmark of ALI. CS increases EC permeability in vitro and in vivo; however, the underlying mechanism is not well understood. In this study, we found that only 6 h of exposure to CS impaired endothelial barrier function in vivo, an effect associated with increased oxidative stress in the lungs and attenuated by the antioxidant N-acetylcysteine (NAC). CS also exacerbated lipopolysaccharide (LPS)-induced increase in vascular permeability in vivo. Similar additive effects were also seen in cultured lung EC exposed to cigarette smoke extract (CSE) and LPS. We further demonstrated that CSE caused disruption of focal adhesion complexes (FAC), F-actin fibers, and adherens junctions (AJ) and decreased activities of RhoA and focal adhesion kinase (FAK) in cultured lung EC. CSE-induced inhibition of RhoA and FAK, endothelial barrier dysfunction, and disassembly of FAC, F-actin, and AJ were prevented by NAC. In addition, the deleterious effects of CSE on FAC, F-actin fibers, and AJ were blunted by overexpression of constitutively active RhoA and of FAK. Our data indicate that CS causes endothelial barrier dysfunction via oxidative stress-mediated inhibition of RhoA and FAK.

Differential expression of MMP-2 and -9 and their inhibitors in fetal lung cells exposed to mechanical stretch: regulation by IL-10.

STUDY OBJECTIVES: Abnormal remodeling of the extracellular matrix (ECM) has been implicated in the pathogenesis of bronchopulmonary dysplasia. However, the contribution of lung parenchymal cells to ECM remodeling after mechanical injury is not well defined. The objective of these studies was to investigate in vitro the release of MMP-2 and -9 and their respective inhibitors TIMP-2 and -1, and to explore potential regulation by IL-10. DESIGN: Mouse fetal epithelial cells and fibroblasts isolated on E18-19 of gestation were exposed to 20% cyclic stretch to simulate lung injury. MMP-2 and MMP-9 activity were investigated by zymography and ELISA. TIMP-1 and TIMP-2 abundance were analyzed by Western blot. RESULTS: We found that mechanical stretch increased MMP-2 and decreased TIMP-2 in fibroblasts, indicating that excessive stretch promotes MMP-2 activation, expressed as the MMP-2/TIMP-2 ratio. Incubation with IL-10 did not change MMP-2 activity. In contrast, mechanical stretch of epithelial cells decreased MMP-9 activity and the MMP-9/TIMP-1 ratio by 60-70%. When IL-10 was added, mechanical stretch increased the MMP-9/TIMP-1 ratio by 50%. CONCLUSIONS: We conclude that mechanical stretch differentially affects MMP-2/9 and their inhibitors in fetal lung cells. IL-10 modulates MMP-9 activity through a combination of effects on MMP-9 and TIMP-1 levels.

IL-10 inhibits inflammatory cytokines released by fetal mouse lung fibroblasts exposed to mechanical stretch.

BACKGROUND: Mechanical ventilation plays an important role in the pathogenesis of bronchopulmonary dysplasia. However, the molecular mechanisms by which excessive stretch induces lung inflammation are not well characterized. OBJECTIVES: In this study, we investigated in vitro the contribution of lung mesenchymal cells to the inflammatory response mediated by mechanical stretch and the potential protective role of IL-10. METHODS: Fetal mouse lung fibroblasts isolated during the saccular stage of lung development were exposed to 20% cyclic stretch to simulate mechanical injury. The phenotype of cultured fibroblasts was investigated by red oil O and alpha-smooth muscle actin (α-SMA) staining. Cell necrosis, apoptosis, and inflammation were analyzed by lactate dehydrogenase release, cleaved caspase-3 activation and release of cytokines and chemokines into the supernatant, respectively. RESULTS: First, we characterized the phenotype of the cultured fibroblasts and found an absence of red oil O staining and 100% positive staining for α-SMA, indicating that cultured fibroblasts were myofibroblasts. Mechanical stretch increased necrosis and apoptosis by two- and three-fold, compared to unstretched samples. Incubation of monolayers with IL-10 prior to stretch did not affect necrosis but significantly decreased apoptosis. Mechanical stretch increased release of pro-inflammatory cytokines and chemokines IL-1ß, MCP-1, RANTES, IL-6, KC and TNF-α into the supernatant by 1.5- to 2.5-fold, and administration of IL-10 before stretch blocked that release. CONCLUSIONS: Our data demonstrate that lung interstitial cells may play a significant role in the inflammatory cascade triggered by mechanical stretch. IL-10 protects fetal fibroblasts from injury secondary to stretch. Pediatr. Pulmonol. 2011; 46:640-649. © 2011 Wiley-Liss, Inc.

A role for caveolin-1 in mechanotransduction of fetal type II epithelial cells.

Mechanical forces are critical for fetal lung development. Using surfactant protein C (SP-C) as a marker, we previously showed that stretch-induced fetal type II cell differentiation is mediated via the ERK pathway. Caveolin-1, a major component of the plasma membrane microdomains, is important as a signaling protein in blood vessels exposed to shear stress. Its potential role in mechanotransduction during fetal lung development is unknown. Caveolin-1 is a marker of type I epithelial cell phenotype. In this study, using immunocytochemistry, Western blotting, and immunogold electron microscopy, we first demonstrated the presence of caveolin-1 in embryonic day 19 (E19) rat fetal type II epithelial cells. By detergent-free purification of lipid raft-rich membrane fractions and fluorescence immunocytochemistry, we found that mechanical stretch translocates caveolin-1 from the plasma membrane to the cytoplasm. Disruption of the lipid rafts with cholesterol-chelating agents further increased stretch-induced ERK activation and SP-C gene expression compared with stretch samples without disruptors. Similar results were obtained when caveolin-1 gene was knocked down by small interference RNA. In contrast, adenovirus overexpression of the wild-type caveolin-1 or delivery of caveolin-1 scaffolding domain peptide inside the cells decreased stretch-induced ERK phosphorylation and SP-C mRNA expression. In conclusion, our data suggest that caveolin-1 is present in E19 fetal type II epithelial cells. Caveolin-1 is translocated from the plasma membrane to the cytoplasm by mechanical stretch and functions as an inhibitory protein in stretch-induced type II cell differentiation via the ERK pathway.

Mechanical stretch promotes fetal type II epithelial cell differentiation via shedding of HB-EGF and TGF-alpha.

The mechanisms by which mechanical forces promote fetal lung development are not fully understood. Here, we investigated differentiation of fetal type II epithelial cells via the epidermal growth factor receptor (EGFR) in response to mechanical strain. First, we showed that incubation of embryonic day (E) 19 fetal type II cells with recombinant heparin-binding EGF-like growth factor (HB-EGF) or transforming growth factor (TGF)-alpha, but not with amphiregulin (AR), betacellulin (BTC) or epiregulin (EPR), increased fetal type II cell differentiation, as measured by surfactant protein B/C mRNA and protein levels. Next, we demonstrated that 5% cyclic stretch of E19 monolayers transfected with plasmid encoding alkaline phosphatase (AP)-tagged ligands shed mature HB-EGF and TGF-alpha into the supernatant and promoted type II cell differentiation. Release of these ligands was also observed in E19 cells subjected to higher degrees of cyclic strain, but not in cells exposed to continuous stretch. Interestingly, the addition of fibroblasts to type II cell cultures did not enhance release of HB-EGF. Whereas HB-EGF shedding was also detected in E18 cells exposed to 5% cyclic stretch, release of this ligand after 2.5% sustained stretch was restricted to cells isolated on E18 of gestation. In addition, mechanical stretch released EGF, AR and BTC. We conclude that mechanical stretch promotes fetal type II cell differentiation via ectodomain shedding of HB-EGF and TGF-alpha. The magnitude of shedding varied depending on gestational age, ligand, and strain protocol. These studies provide novel mechanistic information potentially relevant to fetal lung development and to mechanical ventilation-induced lung injury.

Roles of RhoA and Rac1 on actin remodeling and cell alignment and differentiation in fetal type II epithelial cells exposed to cyclic mechanical stretch.

Mechanical forces are critical for normal fetal lung development. However, the signaling events that promote lung maturation are not fully understood. In this study, the authors analyzed the role of Rho family guanidine triphosphatases (GTPases) in isolated embryonic day 19 (E19) fetal type II epithelial cells exposed to 5% cyclic stretch. The results showed that mechanical strain stimulated RhoA within 5 minutes of initiation of force. Rac1 was also activated, but not Cdc42. After 6 hours of equibiaxial stretch, actin filaments were oriented parallel to the long axis of the cells. By 16 hours, actin fibers still maintained the same orientation, but their intensity decreased when compared to 6 hours. These findings temporally correlated with a decrease in RhoA stimulation. Using adenoviruses encoding dominant negative mutants of RhoA and Rac1, the authors observed that both GTPases are important for strain-induced stress fiber formation, cell alignment, and extracellular signal-regulated kinase (ERK) phosphorylation. However, whereas inhibition of Rho increased surfactant protein C (SP-C) mRNA expression (a marker of type II cell differentiation), suppression of Rac had no effects. These studies suggest that RhoA and Rac1 regulate actin remodeling and cell alignment in fetal type II cells exposed to mechanical stretch. RhoA is a negative regulator of stretch-induced type II cell maturation.

Interleukin-10 protects cultured fetal rat type II epithelial cells from injury induced by mechanical stretch.

Mechanical ventilation plays a central role in the pathogenesis of bronchopulmonary dysplasia. However, the mechanisms by which excessive stretch of fetal or neonatal type II epithelial cells contributes to lung injury are not well defined. In these investigations, isolated embryonic day 19 fetal rat type II epithelial cells were cultured on substrates coated with fibronectin and exposed to 5% or 20% cyclic stretch to simulate mechanical forces during lung development or lung injury, respectively. Twenty percent stretch of fetal type II epithelial cells increased necrosis, apoptosis, and proliferation compared with control, unstretched samples. By ELISA and real-time PCR (qRT-PCR), 20% stretch increased secretion of IL-8 into the media and IL-8 gene expression and inhibited IL-10 release. Interestingly, administration of recombinant IL-10 before 20% stretch did not affect cell lysis but significantly reduced apoptosis and IL-8 release compared with stretched samples without IL-10. Collectively, our studies suggest that IL-10 may play an important role in protection of fetal type II epithelial cells from injury secondary to stretch.

DNA microarray reveals novel genes induced by mechanical forces in fetal lung type II epithelial cells.

Mechanical forces are essential for normal fetal lung development. However, the cellular and molecular mechanisms regulating this process are still poorly defined. In this study, we used oligonucleotide microarrays to investigate gene expression in cultured embryonic d 19 rat fetal lung type II epithelial cells exposed to a level of mechanical strain similar to the developing lung. Significance Analysis of Microarrays (SAM) identified 92 genes differentially expressed by strain. Interestingly, several members of the solute carrier family of amino acid transporter (Slc7a1, Slc7a3, Slc6a9, and tumor-associated protein 1) genes involved in amino acid synthesis (Phgdh, Psat1, Psph, Cars, and Asns), as well as the amiloride-sensitive epithelial sodium channel gene (Scnn1a) were up-regulated by the application of force. These results were confirmed by quantitative real-time PCR (qRT-PCR). Thus, this study identifies genes induced by strain that may be important for amino acid signaling pathways and protein synthesis in fetal type II cells. In addition, these data suggest that mechanical forces may contribute to facilitate lung fluid reabsorption in preparation for birth. Taken together, the present investigation provides further insights into how mechanical forces may modulate fetal lung development.