Comprehensive epigenomic profiling of human alveolar epithelial differentiation identifies key epigenetic states and transcription factor co-regulatory networks for maintenance of distal lung identity.

BACKGROUND: Disruption of alveolar epithelial cell (AEC) differentiation is implicated in distal lung diseases such as chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, and lung adenocarcinoma that impact morbidity and mortality worldwide. Elucidating underlying disease pathogenesis requires a mechanistic molecular understanding of AEC differentiation. Previous studies have focused on changes of individual transcription factors, and to date no study has comprehensively characterized the dynamic, global epigenomic alterations that facilitate this critical differentiation process in humans. RESULTS: We comprehensively profiled the epigenomic states of human AECs during type 2 to type 1-like cell differentiation, including the methylome and chromatin functional domains, and integrated this with transcriptome-wide RNA expression data. Enhancer regions were drastically altered during AEC differentiation. Transcription factor binding analysis within enhancer regions revealed diverse interactive networks with enrichment for many transcription factors, including NKX2-1 and FOXA family members, as well as transcription factors with less well characterized roles in AEC differentiation, such as members of the MEF2, TEAD, and AP1 families. Additionally, associations among transcription factors changed during differentiation, implicating a complex network of heterotrimeric complex switching in driving differentiation. Integration of AEC enhancer states with the catalog of enhancer elements in the Roadmap Epigenomics Mapping Consortium and Encyclopedia of DNA Elements (ENCODE) revealed that AECs have similar epigenomic structures to other profiled epithelial cell types, including human mammary epithelial cells (HMECs), with NKX2-1 serving as a distinguishing feature of distal lung differentiation. CONCLUSIONS: Enhancer regions are hotspots of epigenomic alteration that regulate AEC differentiation. Furthermore, the differentiation process is regulated by dynamic networks of transcription factors acting in concert, rather than individually. These findings provide a roadmap for understanding the relationship between disruption of the epigenetic state during AEC differentiation and development of lung diseases that may be therapeutically amenable.

Wnt/ß-catenin activation and macrophage induction during liver cancer development following steatosis.

Obesity confers an independent risk for carcinogenesis. In the liver, steatosis often proceeds cancer formation; however, the mechanisms by which steatosis promotes carcinogenesis is unknown. We hypothesize that steatosis alters the microenvironment to promote proliferation of tumor initiating cells (TICs) and carcinogenesis. We used several liver cancer models to address the mechanisms underlying the role of obesity in cancer and verified these findings in patient populations. Using bioinformatics analysis and verified by biochemical assays, we identified that hepatosteatosis resulting from either Pten deletion or transgenic expression of HCV core/NS5A proteins, promotes the activation of Wnt/ß-catenin. We verified that high fat diet lipid accumulation is also capable of inducing Wnt/ß-catenin. Caloric restriction inhibits hepatosteatosis, reduces Wnt/ß-catenin activation and blocks the expansion of TICs leading to complete inhibition of tumorigenesis without affecting the phosphatase and tensin homologue deleted on chromosome 10 (PTEN) loss regulated protein kinase B (AKT) activation. Pharmacological inhibition or loss of the Wnt/ß-catenin signal represses TIC growth in vitro, and decreases the accumulation of TICs in vivo. In human liver cancers, ontology analysis of gene set enrichment analysis (GSEA)-defined Wnt signature genes indicates that Wnt signaling is significantly induced in tumor samples compared with healthy livers. Indeed, Wnt signature genes predict 90% of tumors in a cohort of 558 patient samples. Selective depletion of macrophages leads to reduction of Wnt and suppresses tumor development, suggesting infiltrating macrophages as a key source for steatosis-induced Wnt expression. These data established Wnt/ß-catenin as a novel signal produced by infiltrating macrophages induced by steatosis that promotes growth of tumor progenitor cells, underlying the increased risk of liver tumor development in obese individuals.

Transforming Growth Factor-ß1 Induced Epithelial Mesenchymal Transition is blocked by a chemical antagonist of translation factor eIF4E.

The epithelial to mesenchymal transition (EMT) imparts disease-defining properties to epithelial cells in cancer and organ fibrosis. Prior studies identify EMT control points at the level of transcription and translation, and indicate that activation of translation initiation factor 4E (eIF4E) is involved in the mechanisms coordinating these two levels of control. Here we show that 4Ei-1, a specific chemical antagonist of the eIF4E-mRNA cap interaction, potently inhibits transforming growth factor beta 1 (TGF-ß1) mediated EMT in lung epithelial cells. Upon treatment with TGF-ß1, we observed a rapid recruitment of Snail1 mRNA into the actively translated polysome pool accompanied by accumulation of the EMT transcription factor Snail1 in the nucleus. 4Ei-1 blocks ribosome recruitment to the Snail1 transcript thereby preventing accumulation of the Snail1 protein in the nucleus. Our findings establish an obligatory role for upstream translational control of downstream Snail1-mediated transcriptional events in TGF-ß1 induced EMT, and provide proof of concept for efforts to pharmacologically modulate the eIF4E-cap interaction as a means to inhibit pathological EMT in the setting of cancer and organ fibrosis.

Dynamic involvement of ATG5 in cellular stress responses.

Autophagy maintains cell and tissue homeostasis through catabolic degradation. To better delineate the in vivo function for autophagy in adaptive responses to tissue injury, we examined the impact of compromised autophagy in mouse submandibular glands (SMGs) subjected to main excretory duct ligation. Blocking outflow from exocrine glands causes glandular atrophy by increased ductal pressure. Atg5(f/-);Aqp5-Cre mice with salivary acinar-specific knockout (KO) of autophagy essential gene Atg5 were generated. While duct ligation induced autophagy and the expression of inflammatory mediators, SMGs in Atg5(f/-);Aqp5-Cre mice, before ligation, already expressed higher levels of proinflammatory cytokine and Cdkn1a/p21 messages. Extended ligation period resulted in the caspase-3 activation and acinar cell death, which was delayed by Atg5 knockout. Moreover, expression of a set of senescence-associated secretory phenotype (SASP) factors was elevated in the post-ligated glands. Dysregulation of cell-cycle inhibitor CDKN1A/p21 and activation of senescence-associated ß-galactosidase were detected in the stressed SMG duct cells. These senescence markers peaked at day 3 after ligation and partially resolved by day 7 in post-ligated SMGs of wild-type (WT) mice, but not in KO mice. The role of autophagy-related 5 (ATG5)-dependent autophagy in regulating the tempo, duration and magnitude of cellular stress responses in vivo was corroborated by in vitro studies using MEFs lacking ATG5 or autophagy-related 7 (ATG7) and autophagy inhibitors. Collectively, our results highlight the role of ATG5 in the dynamic regulation of ligation-induced cellular senescence and apoptosis, and suggest the involvement of autophagy resolution in salivary repair.

Deletion of ATG5 shows a role of autophagy in salivary homeostatic control.

Autophagy is a catabolic pathway utilized to maintain a balance among the synthesis, degradation, and recycling of cellular components, thereby playing a role in cell growth, development, and homeostasis. Previous studies revealed that a conditional knockout of essential member(s) of autophagy in a variety of tissues causes changes in structure and function of these tissues. Acinar cell-specific expression of knocked-in Cre recombinase through control of aquaporin 5 (Aqp5) promoter/enhancer (Aqp5-Cre) allows us to specifically inactivate Atg5, a protein necessary for autophagy, in salivary acinar cells of Atg5(f/f);Aqp5-Cre mice. There was no difference in apoptotic or proliferation levels in salivary glands of Atg5/Cre mice from each genotype. However, H&E staining and electron microscopy studies revealed modestly enlarged acinar cells and accumulated secretory granules in salivary glands of Atg5(f/f);Aqp5-Cre mice. Salivary flow rates and amylase contents of Atg5/Cre mice indicated that acinar-specific inactivation of ATG5 did not alter carbachol-evoked saliva and amylase secretion. Conversely, autophagy intersected with salivary morphological and secretory manifestations induced by isoproterenol administration. These results identified a role for autophagy as a homeostasis control in salivary glands. Collectively, Atg5(f/f);Aqp5-Cre mice would be a useful tool to enhance our understanding of autophagy in adaptive responses following targeted head and neck radiation or Sjögren syndrome.

N-acetylcysteine inhibits alveolar epithelial-mesenchymal transition.

The ability of transforming growth factor-beta1 (TGF-beta1) to induce epithelial-mesenchymal transition (EMT) in alveolar epithelial cells (AEC) in vitro and in vivo, together with the demonstration of EMT in biopsies of idiopathic pulmonary fibrosis (IPF) patients, suggests a role for TGF-beta1-induced EMT in disease pathogenesis. We investigated the effects of N-acetylcysteine (NAC) on TGF-beta1-induced EMT in a rat epithelial cell line (RLE-6TN) and in primary rat alveolar epithelial cells (AEC). RLE-6TN cells exposed to TGF-beta1 for 5 days underwent EMT as evidenced by acquisition of a fibroblast-like morphology, downregulation of the epithelial-specific protein zonula occludens-1, and induction of the mesenchymal-specific proteins alpha-smooth muscle actin (alpha-SMA) and vimentin. These changes were inhibited by NAC, which also prevented Smad3 phosphorylation. Similarly, primary alveolar epithelial type II cells exposed to TGF-beta1 also underwent EMT that was prevented by NAC. TGF-beta1 decreased cellular GSH levels by 50-80%, whereas NAC restored them to approximately 150% of those found in TGF-beta1-treated cells. Treatment with glutathione monoethyl ester similarly prevented an increase in mesenchymal marker expression. Consistent with its role as an antioxidant and cellular redox stabilizer, NAC dramatically reduced intracellular reactive oxygen species production in the presence of TGF-beta1. Finally, inhibition of intracellular ROS generation during TGF-beta1 treatment prevented alveolar EMT, but treatment with H2O2 alone did not induce EMT. We conclude that NAC prevents EMT in AEC in vitro, at least in part through replenishment of intracellular GSH stores and limitation of TGF-beta1-induced intracellular ROS generation. We speculate that beneficial effects of NAC on pulmonary function in IPF may be mediated by inhibitory effects on alveolar EMT.

Vesicular stomatitis virus G-pseudotyped lentivirus vectors mediate efficient apical transduction of polarized quiescent primary alveolar epithelial cells.

We investigated the use of lentivirus vectors for gene transfer to quiescent alveolar epithelial cells. Primary rat alveolar epithelial cells (AEC) grown on plastic or as polarized monolayers on tissue culture-treated polycarbonate semipermeable supports were transduced with a replication-defective human immunodeficiency virus-based lentivirus vector pseudotyped with the vesicular stomatitis virus G (VSV-G) protein and encoding an enhanced green fluorescent protein reporter gene. Transduction efficiency, evaluated by confocal microscopy and quantified by fluorescence-activated cell sorting, was dependent on the dose of vector, ranging from 4% at a multiplicity of infection (MOI) of 0.1 to 99% at an MOI of 50 for AEC grown on plastic. At a comparable titer and MOI, transduction of these cells by a similarly pseudotyped murine leukemia virus vector was approximately 30-fold less than by the lentivirus vector. Importantly, comparison of lentivirus-mediated gene transfer from the apical or basolateral surface of confluent AEC monolayers (R(t) > 2 kOmega. cm(2); MOI = 10) revealed efficient transduction only when VSV-G-pseudotyped lentivirus was applied apically. Furthermore, treatment with EGTA to increase access to the basolateral surface did not increase transduction of apically applied virus, indicating that transduction was primarily via the apical membrane domain. In contrast, differentiated tracheal epithelial cells were transduced by apically applied lentivirus only in the presence of EGTA and at a much lower overall efficiency (approximately 15-fold) than was observed for AEC. Efficient transduction of AEC from the apical cell surface supports the feasibility of using VSV-G-pseudotyped lentivirus vectors for gene transfer to the alveolar epithelium and suggests that differences exist between upper and lower airways in the polarity of available receptors for the VSV-G protein.

Re-evaluating the Na(+) conductance of adult rat alveolar type II pneumocytes: evidence for the involvement of cGMP-activated cation channels.

1. Alveolar epithelial type II pneumocytes were isolated and purified from adult rat lung by elastase digestion and differential adhesion, and cultured in serum-free medium for approximately 2 days on glass coverslips for subsequent patch-clamp studies employing symmetrical sodium isethionate solutions. 2. Whole-cell Na(+) currents exhibited essentially linear current-voltage relationships which were mildly inhibited (by approximately 25 %) by 10 microM amiloride. In contrast, 1 mM Zn(2+) inhibited the currents by approximately 55 % with an IC(50) of approximately 134 microM and maximal blockade achieved between 5 and 10 mM. The effects of Zn(2+) and amiloride were additive, and independent of the order of blocker addition. 3. Gd(2+), Zn(2+) and La(3+) at 10 mM were all effective at rapidly, reversibly and significantly blocking the amiloride-insensitive currents by approximately 60%. in contrast, Ni(2+) was a very weak inhibitor (30 % inhibition at 10 mM). 4. Pimozide (10 microM) caused inhibition of whole-cell cation conductance by approximately 55 %. The inhibitory effect of pimozide was concentration dependent with an IC(50) of approximately 1 microM and was maximally effective between 10 and 30 microM. Sequential addition of Zn(2+) and pimozide, in either order, revealed no overlapping inhibitory effect on the amiloride-insensitive conductance, and supported the notion that the Zn(2+)- and pimozide-sensitive currents are identical. 5. The amiloride-insensitive, Zn(2+)-blockable conductance was characterised by a Na(+)/K(+) permeability ratio (P(Na)/P(K)) of 0.73 +/- 0.02. 6. 8Br-cGMP (100 microM), a membrane-permeable analogue of cGMP, evoked a robust activation of whole-cell cation conductance to 220 % of control. This activation was apparent in either the absence or the presence of 10 microM amiloride, but was completely abolished in the presence of Zn(2+). 7. These data support the in vivo and in situ observations of a substantial amiloride-resistant Na(+) conductance, demonstrate directly that cyclic nucleotide-gated non-selective cation channels are functionally expressed in alveolar epithelial type II cells, and suggest that these channels may contribute to the fluid-reabsorptive driving force in adult lung.

Extracellular glutathione inhibits oxygen-induced permeability changes in alveolar epithelial monolayers.

Exposure to high fractional inspired oxygen for 24 h increases permeability of the alveolar epithelium, contributing to the clinical manifestations of oxygen toxicity. Utilizing a model of the alveolar epithelium in which isolated rat type II cells form polarized monolayers on polycarbonate filters [transepithelial resistance (R(t)) > 1 k Omega x cm(2) by day 4], we evaluated the ability of reduced glutathione (GSH) to ameliorate these changes. On day 4, apical fluid was replaced with culture medium containing 1) no additives, 2) GSH (500 microM), or 3) GSH (500 microM) + glutathione reductase (0.5 U/ml) + nicotinamide adenine dinucleotide phosphate (250 microM). Monolayers were exposed (for 24 h) to room air (control) or 95% O(2), each containing 5% CO(2). After 24 h of hyperoxia, R(t) for condition 1 decreased by 45% compared with control (P < 0.001). In conditions 2 and 3, R(t) did not decrease significantly (P = not significant). Hyperoxia-induced decreases in active ion transport were observed for conditions 1 and 2 (P < 0.05), but not for condition 3 (P = not significant). These findings indicate that extracellular GSH may protect the alveolar epithelium against hyperoxia-induced injury. Addition of glutathione reductase and nicotinamide adenine dinucleotide phosphate may further augment these protective effects of GSH.

4-Hydroxy-2-nonenal increases gamma-glutamylcysteine synthetase gene expression in alveolar epithelial cells.

Previous studies from this laboratory demonstrated that 4-hydroxy-2-nonenal (4HNE), a lipid peroxidation product, induces expression of gamma-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in de novo glutathione (GSH) synthesis, in rat alveolar epithelial L2 cells. The present study demonstrates that 4HNE also induces GCS in primary cultured alveolar epithelial type II (AT2) cells. Enzyme activity, protein content, and messenger RNA levels of both the catalytic (GCS-HS) and regulatory (GCS-LS) subunits were significantly increased in AT2 cells treated with 5 or 10 microM 4HNE, the same concentrations that induced GCS expression in L2 cells. As in L2 cells, 4HNE induced a greater AT2-cell increase in GCS-LS than in GCS-HS, suggesting that modulation of GCS-LS may play a dominant role in regulating GSH concentration in response to oxidative stress. Additional studies using mitogen-activated protein kinase pathway inhibitors showed that induction by 4HNE of GCS-HS, but not GCS-LS, was mediated through activation of the extracellular regulated kinase pathway in L2 cells. The results demonstrate that L2 cells maintain the same responsiveness to oxidant challenge as do primary cultured AT2 cells in terms of increasing GSH synthetic capacity, and that different pathways are involved in the induction of two GCS subunits by 4HNE.

Integrin alpha(3)-subunit expression modulates alveolar epithelial cell monolayer formation.

We investigated expression of the alpha(3)-integrin subunit by rat alveolar epithelial cells (AECs) grown in primary culture as well as the effects of monoclonal antibodies with blocking activity against the alpha(3)-integrin subunit on AEC monolayer formation. alpha(3)-Integrin subunit mRNA and protein were detectable in AECs on day 1 and increased with time in culture. alpha(3)- and beta(1)-integrin subunits coprecipitated in immunoprecipitation experiments with alpha(3)- and beta(1)-subunit-specific antibodies, consistent with their association as the alpha(3)beta(1)-integrin receptor at the cell membrane. Treatment with blocking anti-alpha(3) monoclonal antibody from day 0 delayed development of transepithelial resistance, reduced transepithelial resistance through day 5 compared with that in untreated AECs, and resulted in large subconfluent patches in monolayers viewed by scanning electron microscopy on day 3. These data indicate that alpha(3)- and beta(1)-integrin subunits are expressed in AEC monolayers where they form the heterodimeric alpha(3)beta(1)-integrin receptor at the cell membrane. Blockade of the alpha(3)-integrin subunit inhibits formation of confluent AEC monolayers. We conclude that the alpha(3)-integrin subunit modulates formation of AEC monolayers by virtue of the key role of the alpha(3)beta(1)-integrin receptor in AEC adhesion.

Differential regulation of rat aquaporin-5 promoter/enhancer activities in lung and salivary epithelial cells.

Aquaporin-5 (AQP5) is a water channel protein that is selectively expressed in respiratory, salivary, and lacrimal tissues. In order to establish the tissue-specific transcriptional programs that underlie its lung- and salivary-specific expression, a 4.5-kilobase pair DNA fragment encompassing the 5'-flanking region of the rat AQP5 gene has been characterized in detail. A major transcription start site utilized in lung and salivary glands has been localized downstream of a TATAA-like motif. Transient transfection assays of -4.3- and -1.7-AQP5-luciferase constructs in AQP5-expressing lung (MLE-15) and salivary (Pa-4) cells and nonexpressing fibroblast (NIH3T3) and epithelial (HeLa) cells demonstrate preferential transcriptional enhancement of reporter activities in MLE-15 and Pa-4 cells. Transient transfection assays of a series of 5' --> 3' deletion constructs of -4.3-AQP5-luciferase suggest that a common salivary and lung enhancer is located between nucleotides -274 and -139, and a lung-specific enhancer is located between nucleotides -894 and -710. There is one putative lung-specific repressor located in the region of nucleotides -1003/-894 and a common lung and salivary repressor located at nucleotides -503/-385. Moreover, 3' --> 5' deletions up to -171 and -127 base pairs almost abolish transcriptional activation in salivary and lung cells, respectively. Together, our findings indicate that the combination of enhancer/repressor elements within the proximal 5'-flanking region of rat AQP5 gene dictates its restricted expression in both lung and salivary cells.

Epidermal growth factor regulation in adult rat alveolar type II cells of amiloride-sensitive cation channels.

Using the patch-clamp technique, we studied the effects of epidermal growth factor (EGF) on whole cell and single channel currents in adult rat alveolar epithelial type II cells in primary culture in the presence or absence of EGF for 48 h. In symmetrical sodium isethionate solutions, EGF exposure caused a significant increase in the type II cell whole cell conductance. Amiloride (10 microM) produced approximately 20-30% inhibition of the whole cell conductance in both the presence and absence of EGF, such that EGF caused the magnitude of the amiloride-sensitive component to more than double. Northern analysis showed that alpha-, beta- and gamma-subunits of rat epithelial Na(+) channel (rENaC) steady-state mRNA levels were all significantly decreased by EGF. At the single channel level, all active inside-out patches demonstrated only 25-pS channels that were amiloride sensitive and relatively nonselective for cations (P(Na(+))/P(K(+)) approximately 1.0:0.48). Although the biophysical characteristics (conductance, open-state probability, and selectivity) of the channels from EGF-treated and untreated cells were essentially identical, channel density was increased by EGF; the modal channel per patch was increased from 1 to 2. These findings indicate that EGF increases expression of nonselective, amiloride-sensitive cation channels in adult alveolar epithelial type II cells. The contribution of rENaC to the total EGF-dependent cation current under these conditions is quantitatively less important than that of the nonselective cation channels in these cells.

Glutathione aerosol suppresses lung epithelial surface inflammatory cell-derived oxidants in cystic fibrosis.

Cystic fibrosis (CF) is characterized by accumulation of activated neutrophils and macrophages on the respiratory epithelial surface (RES); these cells release toxic oxidants, which contribute to the marked epithelial derangements seen in CF. These deleterious consequences are magnified, since reduced glutathione (GSH), an antioxidant present in high concentrations in normal respiratory epithelial lining fluid (ELF), is deficient in CF ELF. To evaluate the feasibility of increasing ELF GSH levels and enhancing RES antioxidant protection, GSH aerosol was delivered (600 mg twice daily for 3 days) to seven patients with CF. ELF total, reduced, and oxidized GSH increased (P < 0.05, all compared with before GSH therapy), suggesting adequate RES delivery and utilization of GSH. Phorbol 12-myristate 13-acetate-stimulated superoxide anion (O2-.) release by ELF inflammatory cells decreased after GSH therapy (P < 0.002). This paralleled observations that GSH added in vitro to CF ELF inflammatory cells suppressed O2-. release (P < 0.001). No adverse effects were noted during treatment. Together, these observations demonstrate the feasibility of using GSH aerosol to restore RES oxidant-antioxidant balance in CF and support the rationale for further clinical evaluation.

KGF prevents hyperoxia-induced reduction of active ion transport in alveolar epithelial cells.

We evaluated the effects of acute hyperoxic exposure on alveolar epithelial cell (AEC) active ion transport and on expression of Na+ pump (Na+-K+-ATPase) and rat epithelial Na+ channel subunits. Rat AEC were cultivated in minimal defined serum-free medium (MDSF) on polycarbonate filters. Beginning on day 5, confluent monolayers were exposed to either 95% air-5% CO2 (normoxia) or 95% O2-5% CO2 (hyperoxia) for 48 h. Transepithelial resistance (Rt) and short-circuit current (Isc) were determined before and after exposure. Na+ channel alpha-, beta-, and gamma-subunit and Na+-K+-ATPase alpha1- and beta1-subunit mRNA levels were quantified by Northern analysis. Na+ pump alpha1- and beta1-subunit protein abundance was quantified by Western blotting. After hyperoxic exposure, Isc across AEC monolayers decreased by approximately 60% at 48 h relative to monolayers maintained under normoxic conditions. Na+ channel beta-subunit mRNA expression was reduced by hyperoxia, whereas alpha- and gamma-subunit mRNA expression was unchanged. Na+ pump alpha1-subunit mRNA was unchanged, whereas beta1-subunit mRNA was decreased approximately 80% by hyperoxia in parallel with a reduction in beta1-subunit protein. Because keratinocyte growth factor (KGF) has recently been shown to upregulate AEC active ion transport and expression of Na+-K+-ATPase under normoxic conditions, we assessed the ability of KGF to prevent hyperoxia-induced changes in active ion transport by supplementing medium with KGF (10 ng/ml) from day 2. The presence of KGF prevented the effects of hyperoxia on ion transport (as measured by Isc) relative to normoxic controls. Levels of beta1 mRNA and protein were relatively preserved in monolayers maintained in MDSF and KGF compared with those cultivated in MDSF alone. These results indicate that AEC net active ion transport is decreased after 48 h of hyperoxia, likely as a result of a decrease in the number of functional Na+ pumps per cell. KGF largely prevents this decrease in active ion transport, at least in part, by preserving Na+ pump expression.

Mechanisms of EGF-induced stimulation of sodium reabsorption by alveolar epithelial cells.

We investigated the effects of epidermal growth factor (EGF) on active Na+ absorption by alveolar epithelium. Rat alveolar epithelial cells (AEC) were isolated and cultivated in serum-free medium on tissue culture-treated polycarbonate filters. mRNA for rat epithelial Na+ channel (rENaC) alpha-, beta-, and gamma-subunits and Na+ pump alpha1- and beta1-subunits were detected in day 4 monolayers by Northern analysis and were unchanged in abundance in day 5 monolayers in the absence of EGF. Monolayers cultivated in the presence of EGF (20 ng/ml) for 24 h from day 4 to day 5 showed an increase in both alpha1 and beta1 Na+ pump subunit mRNA but no increase in rENaC subunit mRNA. EGF-treated monolayers showed parallel increases in Na+ pump alpha1- and beta1-subunit protein by immunoblot relative to untreated monolayers. Fixed AEC monolayers demonstrated predominantly membrane-associated immunofluorescent labeling with anti-Na+ pump alpha1- and beta1-subunit antibodies, with increased intensity of cell labeling for both subunits seen at 24 h following exposure to EGF. These changes in Na+ pump mRNA and protein preceded a delayed (>12 h) increase in short-current circuit (measure of active transepithelial Na+ transport) across monolayers treated with EGF compared with untreated monolayers. We conclude that EGF increases active Na+ resorption across AEC monolayers primarily via direct effects on Na+ pump subunit mRNA expression and protein synthesis, leading to increased numbers of functional Na+ pumps in the basolateral membranes.

Modulation of t1alpha expression with alveolar epithelial cell phenotype in vitro.

T1alpha is a recently identified gene expressed in the adult rat lung by alveolar type I (AT1) epithelial cells but not by alveolar type II (AT2) epithelial cells. We evaluated the effects of modulating alveolar epithelial cell (AEC) phenotype in vitro on T1alpha expression using either soluble factors or changes in cell shape to influence phenotype. For studies on the effects of soluble factors on T1alpha expression, rat AT2 cells were grown on polycarbonate filters in serum-free medium (MDSF) or in MDSF supplemented with either bovine serum (BS, 10%), rat serum (RS, 5%), or keratinocyte growth factor (KGF, 10 ng/ml) from either day 0 or day 4 through day 8 in culture. For studies on the effects of cell shape on T1alpha expression, AT2 cells were plated on thick collagen gels in MDSF supplemented with BS. Gels were detached on either day 1 (DG1) or day 4 (DG4) or were left attached until day 8. RNA and protein were harvested at intervals between days 1 and 8 in culture, and T1alpha expression was quantified by Northern and Western blotting, respectively. Expression of T1alpha progressively increases in AEC grown in MDSF +/- BS between day 1 and day 8 in culture, consistent with transition toward an AT1 cell phenotype. Exposure to RS or KGF from day 0 prevents the increase in T1alpha expression on day 8, whereas addition of either factor from day 4 through day 8 reverses the increase. AEC cultured on attached gels express high levels of T1alpha on days 4 and 8. T1alpha expression is markedly inhibited in both DG1 and DG4 cultures, consistent with both inhibition and reversal of the transition toward the AT1 cell phenotype. These results demonstrate that both soluble factors and alterations in cell shape modulate T1alpha expression in parallel with AEC phenotype and provide further support for the concept that transdifferentiation between AT2 and AT1 cell phenotypes is at least partially reversible.

Keratinocyte growth factor modulates alveolar epithelial cell phenotype in vitro: expression of aquaporin 5.

We investigated the role of keratinocyte growth factor (KGF) in regulation of alveolar epithelial cell (AEC) phenotype in vitro. Effects of KGF on cell morphology, expression of surfactant apoproteins A, B, and C (SP-A, -B, and -C), and expression of aquaporin 5 (AQP5), a water channel present in situ on the apical surface of alveolar type I (AT1) cells but not expressed in alveolar type II (AT2) cells, were evaluated in AECs grown in primary culture. Observations were made on AEC monolayers grown in serum-free medium without KGF (control) or grown continuously in the presence of KGF (10 ng/ml) from either Day 0 (i.e., the time of plating) or Day 4 or 6 through Day 8 in culture. AECs monolayers express AQP5 only on their apical surfaces as determined by cell surface biotinylation studies. Control AECs grown in the absence of KGF through Day 8 express increasing levels of AQP5, consistent with transition toward the AT1 cell phenotype. Exposure of AECs to KGF from Day 0 results in decreased AQP5 expression, retention of a cuboidal morphology, and greater numbers of lamellar bodies relative to control on Day 8 in culture. AECs treated with KGF from Day 4 or 6 exhibit a decrease in AQP5 expression through subsequent days in culture, as well as an increase in expression of surfactant apoproteins. These data, showing that KGF both prevents and reverses the increase in AQP5 (and decrease in surfactant apoprotein) expression that accompanies progression of the AT2 toward the AT1 cell phenotype, support the concepts that transdifferentiation between AT2 and AT1 cell phenotypes is at least partially reversible and that KGF may play a major role in modulating AEC phenotype.

Na(+)-K(+)-ATPase expression in alveolar epithelial cells: upregulation of active ion transport by KGF.

We evaluated the effects of keratinocyte growth factor (KGF) on alveolar epithelial cell (AEC) active ion transport and on rat epithelial Na channel (rENaC) subunit and Na(+)-K(+)-adenosinetriphosphatase (ATPase) subunit isoform expression using monolayers of AEC grown in primary culture. Rat alveolar type II cells were plated on polycarbonate filters in serum-free medium, and KGF (10 ng/ml) was added to confluent AEC monolayers on day 4 in culture. Exposure of AEC monolayers to KGF on day 4 resulted in dose-dependent increases in short-circuit current (Isc) compared with controls by day 5, with further increases occurring through day 8. Relative Na(+)-K(+)-ATPase alpha 1-subunit mRNA abundance was increased by 41% on days 6 and 8 after exposure to KGF, whereas alpha 2-subunit mRNA remained only marginally detectable in both the absence and presence of KGF. Levels of mRNA for the beta 1-subunit of Na(+)-K(+)-ATPase did not increase, whereas cellular alpha 1- and beta 1-subunit protein increased 70 and 31%, respectively, on day 6. mRNA for alpha-, beta-, and gamma-rENaC all decreased in abundance after treatment with KGF. These results indicate that KGF upregulates active ion transport across AEC monolayers via a KGF-induced increase in Na pumps, primarily due to increased Na(+)-K(+)-ATPase alpha 1-subunit mRNA expression. We conclude that KGF may enhance alveolar fluid clearance after acute lung injury by upregulating Na pump expression and transepithelial Na transport across the alveolar epithelium.