The rho pathway mediates transition to an alveolar type i cell phenotype during static stretch of alveolar type ii cells

ABSTRACT Stretch is an essential mechanism for lung growth and development. Animal models in which fetal lungs have been chronically over or underdistended demonstrate a disrupted mix of

type II and type I cells, with static overdistention typically promoting a type I cell phenotype. The Rho GTPase family, key regulators of cytoskeletal signaling, are known to mediate

cellular differentiation in response to stretch in other organs. Using a well-described model of alveolar epithelial cell differentiation and a validated stretch device, we investigated the

effects of supraphysiologic stretch on human fetal lung alveolar epithelial cell phenotype. Static stretch applied to epithelial cells suppressed type II cell markers (SP-B and Pepsinogen C,

PGC), and induced type I cell markers (Caveolin-1, Claudin 7 and Plasminogen Activator Inhibitor-1, PAI-1) as predicted. Static stretch was also associated with Rho A activation.

Furthermore, the Rho kinase inhibitor Y27632 decreased Rho A activation and blunted the stretch-induced changes in alveolar epithelial cell marker expression. Together these data provide

further evidence that mechanical stimulation of the cytoskeleton and Rho activation are key upstream events in mechanotransduction-associated alveolar epithelial cell differentiation.

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SIGNALLING CONFERS DIFFERENTIATION PLASTICITY OF AIRWAY PROGENITORS VIA FOSL2 DURING ALVEOLAR REGENERATION Article 02 September 2021 MAIN The alveolar epithelium is composed of two cell

types: type II and type I cells (1). Type II cells are responsible for surfactant production and play a role in lung host defense. Type I cells, although less numerous, cover the majority of

the gas exchange surface area of the lung. Development and maintenance of this mixed population of alveolar epithelial cells depend on both the biochemical milieu of growth factors,

hormones, and extracellular matrix, and the interplay of physical forces mediated intrinsically by the cytoskeleton and extrinsically by cell-cell and cell-matrix interactions. Stretch plays

a critical role in lung development (2). Static stretch provided by fetal lung fluid provides a constant distending force of approximately 2.5 mm Hg (3). Fetal breathing movements provide

intermittent cyclic stretch (4) resulting in 3–5% change in alveolar surface area (3). By comparison, changes in surface area with tidal breathing in adults are minimal (5), whereas

expansion to total lung capacity changes surface area by 40–45% (6). The importance of stretch as a mechanism for lung development has been shown in human pregnancy complicated by premature

membrane rupture (7), in neonatal neuromuscular disorders (8), and in animal models (9). By extension, enhanced stretch, generally from tracheal obstruction, promotes lung growth (10,11),

providing the rationale for the use of tracheal occlusion to reverse pulmonary hypoplasia in congenital diaphragmatic hernia. Although tracheal occlusion increases lung growth through the

retention of fetal lung fluid, the effects of this supraphysiologic stretch on differentiation of the alveolar epithelium are less clear (12). Animal studies suggest that static stretch

favors the formation of type I cells (13), whereas cyclic stretch favors type II cells (14), but the mechanisms by which stretch is translated into molecular signals to modify gene

expression in the alveolar epithelium are poorly understood. The Rho-GTPase family of small messengers is an attractive candidate for mediating stretch-induced cell signaling because of its

tight coupling to the cytoskeleton. As the cytoskeleton is a global receiver and transmitter of mechanical forces (15), Rho-GTP activation could be an early, upstream intracellular event in

response to stretch. Rho GTPases have been implicated in lung branching morphogenesis (16), alveolar epithelial permeability (17), migration (18), and recently, maturation of alveolar type

II cells (19). We now show, using a validated, equibiaxial stretch device and human fetal lung (HFL) epithelial cells, that changes in epithelial cell phenotype between type I and type II

cells with static stretch are associated with activation of the Rho GTPase pathway. METHODS REAGENTS. Dexamethasone, isobutyl methylxanthine (DCI), and 8-bromo-cAMP were purchased from Sigma

Chemical Co. (St. Louis, MO). All other supplies were purchased from Fisher (Fair Lawn, NJ), Pierce (Rockford, IL) or Invitrogen (Carlsbad, CA). Antisera included SP-B (Chemicon, Temecula,

CA), Pepsinogen C (Abcam, Cambridge, UK), Claudin 7 (Zymed, South San Francisco, CA), Plasminogen Activator Inhibitor-1 (BD Transduction Laboratories, Lexington, KY), Caveolin-1α (Santa Cruz

Biotechnologies, Santa Cruz, CA), and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Chemicon). CELL CULTURE. HFLs from 14- to 18-wk therapeutic abortions were obtained from Advance

Bioscience Resources, Inc. (Alameda, CA) and used in protocols approved by the Committee for Human Research at The Children's Hospital of Philadelphia. A stable population of alveolar

type II cells (with an average 10% contaminating fibroblasts, <5% endothelial cells, and no inflammatory cells) were prepared as described previously (20) and plated at a density of 7 ×

105 cells/cm2 on deformable silastic membranes (Specialty Manufacturing, Saginaw, MI) coated with 50 μg/mL of fibronectin (BD Biosciences, Medford, MA) and mounted into custom-made wells.

Waymouth's media containing 10 nM dexamethasone, 0.1 mM 8-bromo-cAMP, and 0.1 mM DCI was used to maintain the type II cell phenotype. EQUIBIAXIAL STRETCH. Cells on silastic membranes

were mounted onto individual cell-stretching devices capable of applying static equibiaxial strain, as described previously (6). Seventy-two hours after plating, cells were stretched

continuously for 24 h at either 10 or 37% change in surface area. These equibiaxial deformations correspond to static stretches in isolated rat lungs at 55 and 100% of total lung capacity,

respectively. CELL VIABILITY AND APOPTOSIS. Ethidium homodimer-1 and calcein AM were added to the wells to assess cell viability (LIVE/DEAD, Molecular Probes, Eugene, OR). Apoptosis was

assessed by immunoblotting for activated caspase 3 (R and D Systems, Minneapolis, MN). Cells treated with 1 μM Staurosporine (Sigma Chemical Co.) served as a positive control. WESTERN

IMMUNOBLOTTING. Cells were harvested in lysis buffer (1% Triton X-100, 150 mM NaCl, 50 mM Tris-HCl, 5 mM ethylenediamine tetraacetic acid, 5% glycerol, pH 8.0) with 1× Protease inhibitor

(Roche, Indianapolis, IN), and samples immunoblotted using NuPAGE Bis-Tris gels (Invitrogen). Primary antibody concentrations were SP-B 1:4000; PGC 1:5000; Claudin 7, PAI-1, and Caveolin 1

at 1:1000; GAPDH at 1:20,000. Secondary antibodies conjugated to Alexa Fluor 680 (Molecular Probes) or IRdye 800 (Rockland, Gilbertsville, PA) were used at a dilution of 1:10,000. Membranes

were analyzed using the Odyssey infrared imaging system (Li-Cor, Lincoln, NE). REAL-TIME REVERSE TRANSCRIPTASE PCR. Total cellular RNA was isolated using RNA STAT-60 Reagent (Tel-Test,

Friarswood, TX). Purity was verified by the OD 260:280 ratio and integrity assessed using the Agilent 2100 bioanalyzer system (Agilent, Palo Alto, CA). Real-time (RT) PCR assays using a

singleplex strategy were done using an ABI Prism 7900 system (ABI, Foster City, CA). Details of the two-step protocol have been described previously (21). The primer/probe sets (listed on

the ABI website; available at: http://www.allgenes.com) were SP-B Hs00167036, PGC Hs00160052, claudin 7 Hs00600772, PAI-1 Hs00167155, Cav-1 Hs00184697, and 18s Hs99999901_S1. Standards for

comparison of RT-PCR results were derived from RNA isolated from alveolar type II cells cultured from HFL or from banked frozen adult human lung tissue. IMMUNOFLUORESCENCE IMAGING.

Experimental membranes with adherent cells were mounted on glass slides using Fluoromount (Sigma Chemical Co.). Cells were fixed with 1% paraformaldehyde in phosphate-buffered saline, and

immunostained with Claudin 7 antibody (1:100). Nuclei were counterstained with DAPI. Fluorescence was examined at 20× with an Olympus IX81 microscope and Slidebook 4.2.0 digital microscopy

software (Olympus, San Diego, CA). STRESS FIBER ANALYSIS. Alexa Fluor 549 Phalloidin (Molecular Probes, Eugene, OR) was used to stain the actin cytoskeleton at the end of the stretching

period. Stress fibers were counted in seven random high power fields (60×) per treatment group using Image Pro-Plus software (Version 6.0, MediaCybernetics Inc, Bethesda, MD) to determine

mean stress fiber intensity per cell. RHO INHIBITION. Y27632, a selective Rho kinase (ROCK) inhibitor (Tocris, Ellisville, MO) was added to cells at a concentration of 20 μM/mL, 1 h before

stretch and maintained throughout the experiment. Direct activation of Rho was assessed using the G-Lisa Rho A Activation Assay (Cytoskeleton, Denver, CO), which measures activated Rho-GTP,

per the manufacturer's instructions. STATISTICAL ANALYSIS. Results are expressed as mean ± SE. Analysis of variance (for LIVE/DEAD) and _t_ tests (all other experiments) were performed

with GraphPad Prism 5.0 for Macintosh (GraphPad, San Diego, CA). All protein and RNA studies were normalized with GAPDH and 18S, respectively. RESULTS STATIC STRETCH MODIFIED ALVEOLAR

EPITHELIAL CELL PHENOTYPE WITHOUT CELL TOXICITY. Random field counting (_n_ = 4 experiments) revealed no changes in cell viability because of culture or stretch (before stretch 92.8 ± 2.6%

live cells; no stretch 24 h 91.6 ± 1.2%; 10% stretch 24 h 94.0 ± 1.1%; 37% stretch 24 h 93.8 ± 2.4%; _p_ > 0.05), and there was no evidence of apoptosis after static stretch (Fig. 1).

RT-PCR revealed decreased expression of the type II cell markers SP-B and PGC (Fig. 2_A_) at 10% (SP-B: 61 ± 8% and PGC: 50 ± 5% of unstretched control) and 37% change in surface area (SP-B:

50 ± 5% and PGC: 51 ± 7%; _n_ = 5–7, _p_ < 0.01). These changes were not evident at the protein level (Fig. 2_B_ and _C_). Induction of RNA for the type I cell markers Claudin 7 and

PAI-1 (Fig. 3_A_) occurred at both 10% (Claudin 7: 1.5 ± 0.09-fold and PAI-1: 2.0 ± 0.04-fold _versus_ unstretched) and 37% change in surface area (Claudin 7: 1.8 ± 0.1-fold and PAI-1: 3.4 ±

0.4-fold; _n_ = 3, _p_ < 0.05). There was a modest induction of Caveolin-1 RNA with 10% stretch that did not reach statistical significance (_n_ = 3, _p_ = 0.08). Immunoblotting

demonstrated induction of PAI-1 and Caveolin-1 protein with 37% stretch (Fig. 3_B_ and C; _n_ = 4–5, _p_ < 0.05), and a modest increase in Claudin 7 protein expression with 10% stretch.

We characterized Claudin 7 localization to the plasma membrane as a proxy for alveolar epithelial barrier changes because the silastic membranes precluded traditional permeability testing.

Claudin 7 was distributed throughout the cytoplasm before stretch (Fig. 4_A_) and in unstretched cells (not shown). With static stretch (Fig. 4_B_ and _C_), Claudin 7 immunostaining was more

prominent at the plasma membrane. RHO-GTP FUNCTION IN RESPONSE TO STATIC STRETCH. Stress fibers are longitudinal bundles of contractile actin-myosin filaments resulting from activation of

the Rho-GTP/ROCK pathway (22). Stress fibers (as shown in Fig. 5_A_) are present in most cultured cells but are markedly increased by stretch (23). There was a 27% increase in the mean

intensity of phalloidin-positive stress fibers per cell at 37% stretch compared with unstretched controls (Fig. 5_B_; _n_ = 3, _p_ < 0.05), whereas no significant change was observed at

10% stretch. Because stress fibers are a late endpoint in Rho pathway activation, we measured direct Rho/ROCK pathway activation by ELISA in response to 37% stretch (Fig. 6). Activated

Rho-GTP increased by 21.4 ± 1.5% 15 min after initiation of 37% stretch (_n_ = 3–4, _p_ < 0.01), followed by a decrease at 1 and 4 h (at 4 h: 46.7 ± 5.4%, _n_ = 4, _p_ < 0.01), with

variable rebound to baseline by 24 h. INHIBITION OF RHO DIMINISHES STRETCH PHENOTYPE CHANGES. To determine whether the stretch induced changes in gene expression could be due to activation

of Rho, we used a selective Rho inhibitor, Y27632, during epithelial cell stretch (Fig. 7). Y27632 partially restored SP-B RNA expression at 10% stretch but not at 37% stretch (_n_ = 4, _p_

< 0.05 _versus_ 10% stretch without inhibitor). By comparison, expression of PAI-1 was blunted at both 10% and 37% stretch in the presence of Y27632 (_n_ = 4–5, _p_ < 0.05 _versus_ no

inhibitor). DISCUSSION Mechanical forces are important regulators of organogenesis and differentiation during fetal development. Although reduced stretch results in lung hypoplasia while

overdistention stimulates lung growth (3), the effects of stretch on differentiation have been less clear. Animal models suggest that static stretch promotes type I cell phenotype (13), and

cyclic stretch promotes type II cell phenotype (24). More recent studies of animal models of congenital diaphragmatic hernia and postmortem human studies suggest that despite improved lung

growth, supraphysiologic stretch _in utero_ does not result in a mature alveolar epithelium with an appropriate mixed population of type I and type II cells (25,26). This is the first study

to assess the impact of supraphysiologic stretch on alveolar epithelial cell differentiation using a well-characterized model of type II cells derived from HFL in which both the local

effects of static stretch and alterations in intracellular pathways can be easily monitored. Mechanistic studies of stretch-induced epithelial differentiation have been hampered by

controversy surrounding alveolar epithelial cell lineage and alveolar epithelial marker expression. The classic dogma—alveolar type II cells as the progenitor for terminally differentiated

alveolar type I cells—came from interpretations of older studies using electron microscopy to understand the resolution of lung injury (27). These early studies were supported by later

evidence that type II cells served as progenitors for injured type I cells in mature lung (28) and that isolated type II cells in culture for over 24 h to lose characteristics of type II

cells and adopt features of type I cells in the absence of serum (29). Another obstacle to understanding the impact of stretch on differentiation is the paucity of markers that clearly

differentiate type I from type II cells across species. Markers such as T1α (30) and RTI40 (31), now recognized as podoplanin, have not been useful in human lung. Markers that clearly

distinguish type I cells from type II cells, such as Caveolin 1, are also found in other cells locally in the lung (32). Antibodies to markers in rodent models have poor crossreactivity to

human cells. This study attempts to solve these problems by using a relevant human model and choosing markers that have been reproducible in this and other human models. Our

well-characterized cell culture model has been used to examine the cell biology and biochemistry of alveolar type I and II cells (20,33–35). Cells are derived from HFL making this model

particularly relevant to lung development. The primary cultures have been consistently ∼90% pure, with contamination chiefly from fibroblasts. However, the use of a human cell culture model

restricts the choice of available type I cell markers. We have previously shown that PAI-1, Caveolin 1, and Claudin 7 are robust, reproducible markers of type I cells in this system (34).

Although not exclusively expressed by type I cells in the lung, they are clearly not expressed by human type II cells or by contaminating fibroblasts. Therefore, our choice of cell culture

system and markers provide a robust model to study the effects of stretch on human fetal alveolar epithelial cell phenotype. Equibiaxial stretch of type II cells had very predictable effects

on the epithelial cell markers. We demonstrated a decline in SP-B and PGC RNA within 24 h of initiating both 10% and 37% stretch, concomitant with an increase in PAI-1 and Caveolin 1 RNA.

Compared with our prior report of epithelial cell marker expression in transdifferentiation experiments (34), the decline in type II cell markers with stretch despite the presence of

hormones was blunted yet significant. In the prior study, DCI withdrawal effectively eliminated SP-B and PGC RNA expression by 96 h, with persistence of some SP-B protein through 120 h and

PGC protein through 96 h. Persistence of type II cell proteins after cessation of RNA expression has been commonly reported in rodent models of alveolar epithelial cell transdifferentiation

(36,37). Importantly, despite culture conditions that should sustain type II cell marker expression, specifically the presence of glucocorticoid and cAMP, stretch significantly impaired SP-B

and PGC expression. Few studies of the effects of stretch on alveolar epithelial cell differentiation have examined type I cell markers (31). In this study, type I cell markers behaved as

predicted, increasing in response to 24 h of static stretch as type II marker expression waned. The magnitude of changes in type I markers was less than we observed by transdifferentiation

previously. Claudin 7 was helpful in this study because of its type I cell specificity specific in HFL (33,34) and its role in alveolar epithelial barrier function (38). Although Claudin 7

behaved similarly to our prior study, demonstrating only a modest induction of RNA and no significant change in protein content over 24 h, it was remarkable that localization of Claudin 7

became directed to the plasma membrane with stretch. Studies of rat type I cell transdifferentiation also observed an increase in Claudin 7 that was maximal after 7d in culture, with a more

plasma membrane distribution in type I cells that correlated with increased barrier function (39). The silastic membranes we used for the static stretch in our system precluded a more

functional approach to assessing barrier function. However, redistribution of Claudin 7 is indirect but consistent evidence of enhanced barrier function with stretch. Taken together, changes

in type I and II cell marker expression clearly show that despite culture conditions that support maintenance of type II cell phenotype, stretch fosters change toward a type I cell

phenotype in HFL epithelial cells. Several signal transduction pathways have been implicated in stretch-mediated lung maturation, including extracellular signal related kinases (24), protein

kinase A and C (2), heparin-binding epidermal growth-like factor, and transforming growth factor alpha (40). The Rho/ROCK pathway is an excellent candidate for transmitting alveolar

epithelial cell stretch into gene expression due to its pivotal role in regulating the actin cytoskeleton, and in mechanotransduction-mediated differentiation, most notably in smooth muscle

cells (41). We provide compelling evidence that Rho pathway activation plays an important role in static stretch-induced differentiation of HFL epithelium: indirectly by the increase in

stress fibers with static stretch and directly by detecting GTP-bound Rho within 15 min of applying static stretch. More importantly, stretch-induced changes in epithelial cell marker

expression were blunted in the presence of a specific Rho inhibitor, Y27632. Our data echo similar observations in rodent type II cells, with the ROCK inhibitor H-1152 blunting the effect of

cyclic stretch on expression of the type II cell marker SP-C (19). Similar to their studies, our data show that Rho A is a negative regulator of type II cell markers, especially at reduced

levels of stretch—5% in their study, and 10% in ours. What is more impressive is that Rho A is a positive regulator of type I cell markers at both levels of stretch in this study, placing

the Rho pathway as a central regulator of alveolar epithelial differentiation in response to stretch. The differences in response between type I and type II markers and between 10 and 37%

stretch may reflect events downstream of Rho A. Y27632 is a ROCK inhibitor, which will account for only one limb of the pathway after Rho A activation leading to inhibition of cofilin and

promoting actin polymerization (23). Because an intact cytoskeleton is a critical factor in mediating mechanotransduction, the role of profilin actions could be important but are not

regulated by ROCK and thus not susceptible to Y27632. The inability of ROCK inhibition to restore type II cell marker expression to unstretched levels illustrates that there are additional

mechanotransduction pathways involved, such as the mitogen-activated protein kinase pathway (24). The resultant intermediate type I/II cell phenotype may simply reflect the short duration of

these experiments. However, they suggest a reasonable explanation for the presence of such intermediate cells observed by others in response to lung injury as repopulating type II cells

begin to transdifferentiate (26,42,43). Although there is increasing evidence of type I to type II cell plasticity (44), it remains unclear whether this is stretch-responsive _in vivo_ and

which pathways might be involved. In summary, we have shown that static stretch is an important determinant of alveolar epithelial cell plasticity and that mechanotransduction is partially

mediated by the Rho pathway. The Rho GTPase pathway may provide an important early indicator of alveolar epithelial cell well being in studies designed to evaluate lung-protective

ventilation strategies and may offer a targeted pathway for the design of novel pharmacologic interventions, due to the accessibility of the alveolar epithelium, to prevent lung injury

during mechanical ventilation. ABBREVIATIONS * DCI: Dexamethasone, isobutyl methylxanthine, and 8-bromo-cAMP * GAPDH: Glyceraldehyde-3-phosphate dehydrogenase * PGC: Pepsinogen C * PAI-1:

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Google Scholar  Download references ACKNOWLEDGEMENTS We thank Ping Wang for cell preparation and James Hayden and Frederick Keeney, from the Wistar Institute Microscopy Facility for

assistance with the stress fiber imaging and image quantitation studies. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Pediatrics, The Children's Hospital of Philadelphia,

University of Pennsylvania School of Medicine, Philadelphia, 19104, PA Cherie D Foster, Linda S Varghese, Linda W Gonzales & Susan H Guttentag * Department of Bioengineering, University

of Pennsylvania School of Engineering and Applied Science, Philadelphia, 19104, PA Susan S Margulies Authors * Cherie D Foster View author publications You can also search for this author

inPubMed Google Scholar * Linda S Varghese View author publications You can also search for this author inPubMed Google Scholar * Linda W Gonzales View author publications You can also

search for this author inPubMed Google Scholar * Susan S Margulies View author publications You can also search for this author inPubMed Google Scholar * Susan H Guttentag View author

publications You can also search for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to Susan H Guttentag. ADDITIONAL INFORMATION Supported by grants HL-077266

(C.D.F.) and HL059959 (S.H.G.) from the National Institutes of Health. RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Foster, C., Varghese, L.,

Gonzales, L. _et al._ The Rho Pathway Mediates Transition to an Alveolar Type I Cell Phenotype During Static Stretch of Alveolar Type II Cells. _Pediatr Res_ 67, 585–590 (2010).

https://doi.org/10.1203/PDR.0b013e3181dbc708 Download citation * Received: 17 September 2009 * Accepted: 17 February 2010 * Issue Date: June 2010 * DOI:

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