Bronchial Epithelial Compression Regulates MAP Kinase Signaling and HB-EGF-like Growth Factor Expression

American Journal of Physiology. Lung Cellular and Molecular Physiology. May, 2002  |  Pubmed ID: 11943653

Airway smooth muscle constriction leads to the development of compressive stress on bronchial epithelial cells. Normal human bronchial epithelial cells exposed to an apical-to-basal transcellular pressure difference equivalent to the computed stress in the airway during bronchoconstriction demonstrate enhanced phosphorylation of extracellular signal-regulated kinase (ERK). The response is pressure dependent and rapid, with phosphorylation increasing 14-fold in 30 min, and selective, since p38 and c-Jun NH(2)-terminal kinase phosphorylation remains unchanged after pressure application. Transcellular pressure also elicits a ninefold increase in expression of mRNA encoding heparin-binding epidermal growth factor-like growth factor (HB-EGF) after 1 h, followed by prominent immunostaining for pro-HB-EGF after 6 h. Inhibition of the ERK pathway with PD-98059 results in a dose-dependent reduction in pressure-induced HB-EGF gene expression. The magnitude of the HB-EGF response to transcellular pressure and tumor necrosis factor (TNF)-alpha (1 ng/ml) is similar, and the combined mechanical and inflammatory stimulus is more effective than either stimulus alone. These results demonstrate that compressive stress is a selective and potent activator of signal transduction and gene expression in bronchial epithelial cells.

Mechanical Stress Triggers Selective Release of Fibrotic Mediators from Bronchial Epithelium

American Journal of Respiratory Cell and Molecular Biology. Feb, 2003  |  Pubmed ID: 12540481

Transforming growth factor-beta (TGF-beta) and endothelin (ET) are found in elevated amounts in the airways of individuals with asthma. The cellular source of these peptides and their role in mediating the airway fibrosis of chronic asthma are unknown. In response to mechanical stresses similar to those occurring in vivo during airway constriction, bronchial epithelial cells increase the steady-state level of mRNA for both ET-1 and ET-2, followed by increased release of ET protein. Mechanical stress also enhances release of TGF-beta2 from a preformed cell-associated pool. TGF-beta2 and ET act individually and, more importantly, synergistically to promote fibrotic protein synthesis in reporter fibroblasts. To confirm the role of these intermediates in stress-induced fibrosis, conditioned medium from mechanically stressed bronchial epithelial cells was shown to elicit fibrotic protein synthesis in reporter fibroblasts; this effect was significantly inhibited by combined treatment with ET receptor antagonists and a neutralizing antibody to TGF-beta2. These data are consistent with a primary pathogenic role for mechanical stress-induced release of both TGF-beta2 and ET in the subepithelial fibrosis that characterizes chronic asthma.

Differentiation-dependent Responsiveness of Bronchial Epithelial Cells to IL-4/13 Stimulation

American Journal of Physiology. Lung Cellular and Molecular Physiology. Jul, 2004  |  Pubmed ID: 15020299

The Th2 cytokines interleukin (IL)-4 and IL-13 are thought to play critical roles in the airway inflammation and hyperresponsiveness that characterize asthma. Recent evidence indicates that IL-13 can mediate these effects by acting directly on airway epithelial cells. Here we evaluated early [signal transducer and activator of transcription (STAT)6 phosphorylation] and delayed [granulocyte/macrophage colony-stimulating factor (GM-CSF) and transforming growth factor-beta(2) (TGF-beta(2)) secretion] responses of airway epithelial cells to IL-4 and IL-13 stimulation and the dependence of these responses on the culture technique employed. As expected, normal human bronchial epithelial cells grown on microporous inserts at an air-liquid interface (ALI) expressed a well-differentiated mucociliary phenotype; in contrast, cells grown on plastic in submerged cultures were poorly differentiated. When stimulated with IL-4 or IL-13, the magnitude and duration of STAT6 phosphorylation under the differing culture conditions were statistically indistinguishable. In contrast, cytokine secretion responses to IL-4 and IL-13 were highly dependent on the culture technique; cells cultured on plastic exhibited significant concentration-dependent increases in GM-CSF and TGF-beta(2) secretion, whereas cells grown at ALI showed no statistically significant response. These results demonstrate that the coupling between early signal transduction responses to IL-4 and IL-13 and downstream functions such as cytokine secretion may be critically dependent on the cell culture technique employed and the resulting differentiation status of bronchial epithelial cells.

Mechanotransduction Through Growth-factor Shedding into the Extracellular Space

Nature. May, 2004  |  Pubmed ID: 15103386

Physical forces elicit biochemical signalling in a diverse array of cells, tissues and organisms, helping to govern fundamental biological processes. Several hypotheses have been advanced that link physical forces to intracellular signalling pathways, but in many cases the molecular mechanisms of mechanotransduction remain elusive. Here we find that compressive stress shrinks the lateral intercellular space surrounding epithelial cells, and triggers cellular signalling via autocrine binding of epidermal growth factor family ligands to the epidermal growth factor receptor. Mathematical analysis predicts that constant rate shedding of autocrine ligands into a collapsing lateral intercellular space leads to increased local ligand concentrations that are sufficient to account for the observed receptor signalling; direct experimental comparison of signalling stimulated by compressive stress versus exogenous soluble ligand supports this prediction. These findings establish a mechanism by which mechanotransduction arises from an autocrine ligand-receptor circuit operating in a dynamically regulated extracellular volume, not requiring induction of force-dependent biochemical processes within the cell or cell membrane.

EGFR Autocrine Signaling in a Compliant Interstitial Space: Mechanotransduction from the Outside In

Cell Cycle (Georgetown, Tex.). Aug, 2004  |  Pubmed ID: 15254423

Cells transduce mechanical forces into biochemical signals; traditionally these processes are thought to occur through direct effects on the cell membrane, the cytoskeleton, or specific transmembrane proteins. In multicellular tissues mechanical forces alter intercellular spacing through redistribution of interstitial fluid. Recent morphological and biochemical observations, bolstered by analytical modeling, support a new paradigm for mechanotransduction arising from constitutive growth factor shedding into a dynamically regulated interstitial volume.

Bronchial Epithelial Compression Regulates Epidermal Growth Factor Receptor Family Ligand Expression in an Autocrine Manner

American Journal of Respiratory Cell and Molecular Biology. May, 2005  |  Pubmed ID: 15705969

The epidermal growth factor receptor (EGFR), an important signaling pathway in airway biology, is stimulated by compressive stress applied to human airway epithelial cells. Although the EGFR ligand, heparin-binding epidermal growth factor-like growth factor (HB-EGF), is known to be released as a result of this stimulation, whether compressive stress enhances expression of other EGFR ligands, and the duration of mechanical compression required to initiate this response, is not known. Human airway epithelial cells were exposed to compressive stress, and expression of four EGFR ligands was examined by quantitative PCR. Cells were exposed to: (1) continuous compressive stress over 8 h, (2) compression with and without EGFR inhibitor (AG1478), or (3) time-limited compression (3.75, 7.5, 15, 30, and 60 min). Compressive stress produced a sustained upregulation of the EGFR ligands HB-EGF, epiregulin, and amphiregulin, but not transforming growth factor-alpha. Inhibition with AG1478 demonstrated that expression of HB-EGF, epiregulin, and amphiregulin is dependent on the signaling via the EGFR. Immunostaining for epiregulin protein demonstrated increased expression with compression and attenuation with EGFR inhibition. The response of all three EGFR ligands persisted long after the mechanical stimulus was removed. Taken together, these data suggest the possibility of a mechanically activated EGFR autocrine feedback loop involving selected EGFR ligands.

Chronic Effects of Mechanical Force on Airways

Annual Review of Physiology. 2006  |  Pubmed ID: 16460284

Airways are embedded in the mechanically dynamic environment of the lung. In utero, this mechanical environment is defined largely by fluid secretion into the developing airway lumen. Clinical, whole lung, and cellular studies demonstrate pivotal roles for mechanical distention in airway morphogenesis and cellular behavior during lung development. In the adult lung, the mechanical environment is defined by a dynamic balance of surface, tissue, and muscle forces. Diseases of the airways modulate both the mechanical stresses to which the airways are exposed as well as the structure and mechanical behavior of the airways. For instance, in asthma, activation of airway smooth muscle abruptly changes the airway size and stress state within the airway wall; asthma also results in profound remodeling of the airway wall. Data now demonstrate that airway epithelial cells, smooth muscle cells, and fibroblasts respond to their mechanical environment. A prominent role has been identified for the epithelium in transducing mechanical stresses, and in both the fetal and mature airways, epithelial cells interact with mesenchymal cells to coordinate remodeling of tissue architecture in response to the mechanical environment.

Computational Modeling of Extracellular Mechanotransduction

Biophysical Journal. Jun, 2006  |  Pubmed ID: 16533844

Mechanotransduction may occur through numerous mechanisms, including potentially through autocrine signaling in a dynamically changing extracellular space. We developed a computational model to analyze how alterations in the geometry of an epithelial lateral intercellular space (LIS) affect the concentrations of constitutively shed ligands inside and below the LIS. The model employs the finite element method to solve for the concentration of ligands based on the governing ligand diffusion-convection equations inside and outside of the LIS, and assumes idealized parallel plate geometry and an impermeable tight junction at the apical surface. Using the model, we examined the temporal relationship between geometric changes and ligand concentration, and the dependence of this relationship on system characteristics such as ligand diffusivity, shedding rate, and rate of deformation. Our results reveal how the kinetics of mechanical deformation can be translated into varying rates of ligand accumulation, a potentially important mechanism for cellular discrimination of varying rate-mechanical processes. Furthermore, our results demonstrate that rapid changes in LIS geometry can transiently increase ligand concentrations in underlying media or tissues, suggesting a mechanism for communication of mechanical state between epithelial and subepithelial cells. These results underscore both the plausibility and complexity of the proposed extracellular mechanotransduction mechanism.

Induction of the Plasminogen Activator System by Mechanical Stimulation of Human Bronchial Epithelial Cells

American Journal of Respiratory Cell and Molecular Biology. Dec, 2006  |  Pubmed ID: 16794260

Mechanical stimulation of the airway epithelium, as would occur during bronchoconstriction, is a potent stimulus and can activate profibrotic pathways. We used DNA microarray technology to examine gene expression in compressed normal human bronchial epithelial cells (NHBE). Compressive stress applied continuously over an 8-h period to NHBE cells led to the upregulation of several families of genes, including a family of plasminogen-related genes that were previously not known to be regulated in this system. Real-time PCR demonstrated a peak increase in gene expression of 8.0-fold for urokinase plasminogen activator (uPA), 16.2-fold for urokinase plasminogen activator receptor (uPAR), 4.2-fold for plasminogen activator inhibitor-1 (PAI-1), and 3.9-fold for tissue plasminogen activator (tPA). Compressive stress also increased uPA protein levels in the cell lysates (112.0 versus 82.0 ng/ml, P = 0.0004), and increased uPA (4.7 versus 3.3 ng/ml, P = 0.02), uPAR (1.3 versus 0.86 ng/ml, P = 0.007), and PAI-1 (50 versus 36 ng/ml, P = 0.006) protein levels in cell culture media. Functional studies demonstrated increased urokinase-dependent plasmin generation in compression-stimulated cells (0.0090 versus 0.0033 OD/min, P = 0.03). In addition, compression led to increased activation of matrix metalloproteinase (MMP)-9 and MMP-2 in a urokinase-dependent manner. In postmortem human lung tissue, we observed an increase in epithelial uPA and uPAR immunostaining in the airways of two patients who died in status asthmaticus compared with minimal immunoreactivity noted in airways from seven lung donors without asthma. Together these observations suggest an integrated response of airway epithelial cells to mechanical stimulation, acting through the plasminogen-activating system to modify the airway microenvironment.

A New Microrheometric Approach Reveals Individual and Cooperative Roles for TGF-beta1 and IL-1beta in Fibroblast-mediated Stiffening of Collagen Gels

FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology. Jul, 2007  |  Pubmed ID: 17341683

The stiffness of the extracellular matrix can profoundly influence cell and tissue behaviors. Thus there is an emerging emphasis on understanding how matrix mechanical environments are established, regulated, and modified. Here we develop a microrheometric assay to measure the mechanical properties of a model extracellular matrix (type I collagen gel) and use it to explore cytokine-induced, cell-mediated changes in matrix mechanical properties. The microrheometric assay uses micron-scale ferrimagnetic beads embedded within collagen gels during fibrillogenesis. The beads are magnetized, then subjected to a twisting field, with the aggregate rotation of the beads measured by a magnetometer. The degree of bead rotation reflects the stiffness of the surrounding matrix. We show that the microscale assay provides stiffness measures for collagen gels comparable to those obtained with standard macroscale rheometry. To demonstrate the utility of the assay for biological discovery, we measure stiffness changes in fibroblast-populated collagen gels exposed to three concentrations of six cytokines over 2 to 14 days. Among the cytokines tested, transforming growth factor-beta1 and interleukin-1beta enhanced matrix stiffness, and together exerted cooperative effects on cellular modulation of matrix mechanics. The microrheometry approach developed here should accelerate the discovery of biological pathways orchestrating cellular modulation of matrix mechanics.

Universal Physical Responses to Stretch in the Living Cell

Nature. May, 2007  |  Pubmed ID: 17538621

With every beat of the heart, inflation of the lung or peristalsis of the gut, cell types of diverse function are subjected to substantial stretch. Stretch is a potent stimulus for growth, differentiation, migration, remodelling and gene expression. Here, we report that in response to transient stretch the cytoskeleton fluidizes in such a way as to define a universal response class. This finding implicates mechanisms mediated not only by specific signalling intermediates, as is usually assumed, but also by non-specific actions of a slowly evolving network of physical forces. These results support the idea that the cell interior is at once a crowded chemical space and a fragile soft material in which the effects of biochemistry, molecular crowding and physical forces are complex and inseparable, yet conspire nonetheless to yield remarkably simple phenomenological laws. These laws seem to be both universal and primitive, and thus comprise a striking intersection between the worlds of cell biology and soft matter physics.

Chronic Intermittent Mechanical Stress Increases MUC5AC Protein Expression

American Journal of Respiratory Cell and Molecular Biology. Oct, 2009  |  Pubmed ID: 19168703

Increased abundance of mucin secretory cells is a characteristic feature of the epithelium in asthma and other chronic airway diseases. We showed previously that the mechanical stresses of airway constriction, both in the intact mouse lung and a cell culture model, activate the epidermal growth factor receptor (EGFR), a known modulator of mucin expression in airway epithelial cells. Here we tested whether chronic, intermittent, short-duration compressive stress (30 cm H(2)O) is sufficient to increase the abundance of MUC5AC-positive cells and intracellular mucin levels in human bronchial epithelial cells cultured at an air-liquid interface. Compressive stress applied for 1 hour per day for 14 days significantly increased the percentage of cells staining positively for MUC5AC protein (22.0 +/- 3.8%, mean +/- SD) relative to unstimulated controls (8.6 +/- 2.6%), and similarly changed intracellular MUC5AC protein levels measured by Western and slot blotting. The effect of compressive stress was gradual, with significant changes in MUC5AC-positive cell numbers evident by Day 7, but required as little as 10 minutes of compressive stress daily. Daily treatment of cells with an EGFR kinase inhibitor (AG1478, 1 muM) significantly but incompletely attenuated the response to compressive stress. Complete attenuation could be accomplished by simultaneous treatment with the combination of AG1478 and a transforming growth factor (TGF)-beta(2) (1 microg/ml)-neutralizing antibody, or with anti-TGF-beta(2) alone. Our findings demonstrate that short duration episodes of mechanical stress, representative of those occurring during bronchoconstriction, are sufficient to increase goblet cell number and MUC5AC protein expression in bronchial epithelial cells in vitro. We propose that the mechanical environment present in asthma may fundamentally bias the composition of airway epithelial lining in favor of mucin secretory cells.

Stretch-induced Mitogen-activated Protein Kinase Activation in Lung Fibroblasts is Independent of Receptor Tyrosine Kinases

American Journal of Respiratory Cell and Molecular Biology. Jul, 2010  |  Pubmed ID: 19684308

Lung growth and remodeling are modulated by mechanical stress, with fibroblasts thought to play a leading role. Little mechanistic information is available about how lung fibroblasts respond to mechanical stress. We exposed cultured lung fibroblasts to tonic stretch and measured changes in phosphorylation status of mitogen-activated protein kinases (MAPKs), selected receptor tyrosine kinases (RTKs), and phospholipase Cgamma1 (PLCgamma1) and activation of the small G-protein Ras. Human lung fibroblasts (LFs) were seeded on matrix-coated silicone membranes and exposed to equibiaxial 10 to 40% static stretch or 20% contraction. LFs were stimulated with EGF, FGF2, or PDGF-BB or exposed to stretch in the presence of inhibitors of EGFR (AG1478), FGFR (PD173074), and PDGFR (AG1296). Phospho-MAPK, phospho-RTK, and phospho-PLCgamma1 levels were measured by Western blotting. Active GTP-Ras was quantified by immunoblotting after pull-down with a glutathione S-transferase-Raf-RBD construct. Normalized p-ERK1/2, p-JNK, and p-p38 levels increased after stretch but not contraction. Ligands to RTKs broadly stimulated MAPKs, with the responses to EGF and PDGF most similar to stretch in terms of magnitude and rank order of MAPK responses. Stretching cells failed to elicit measurable activation of EGFR, FGFR (FRS2alpha phosphorylation), or PDGFR. Potent inhibitors of the kinase activity of each receptor failed to attenuate stretch-induced MAPK activation. PLCgamma1 and Ras, prominent effectors downstream of RTKs, were not activated by stretch. Our findings demonstrate that MAPKs are potently activated by stretch in lung fibroblasts, but, in contrast to stress responses observed in other cell types, RTKs are not necessary for stretch-induced MAPK activation in LFs.

Recent Advances and New Opportunities in Lung Mechanobiology

Journal of Biomechanics. Jan, 2010  |  Pubmed ID: 19804885

Lung function is inextricably linked to mechanics. On short timescales every breath generates dynamic cycles of cell and matrix stretch, along with convection of fluids in the airways and vasculature. Perturbations such airway smooth muscle shortening or surfactant dysfunction rapidly alter respiratory mechanics, with profound influence on lung function. On longer timescales, lung development, maturation, and remodeling all strongly depend on cues from the mechanical environment. Thus mechanics has long played a central role in our developing understanding of lung biology and respiratory physiology. This concise review focuses on progress over the past 5 years in elucidating the molecular origins of lung mechanical behavior, and the cellular signaling events triggered by mechanical perturbations that contribute to lung development, homeostasis, and injury. Special emphasis is placed on the tools and approaches opening new avenues for investigation of lung behavior at integrative cellular and molecular scales. We conclude with a brief summary of selected opportunities and challenges that lie ahead for the lung mechanobiology research community.

An EGFR Autocrine Loop Encodes a Slow-reacting but Dominant Mode of Mechanotransduction in a Polarized Epithelium

FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology. May, 2010  |  Pubmed ID: 20056713

The mechanical landscape in biological systems can be complex and dynamic, with contrasting sustained and fluctuating loads regularly superposed within the same tissue. How resident cells discriminate between these scenarios to respond accordingly remains largely unknown. Here, we show that a step increase in compressive stress of physiological magnitude shrinks the lateral intercellular space between bronchial epithelial cells, but does so with strikingly slow exponential kinetics (time constant approximately 110 s). We confirm that epidermal growth factor (EGF)-family ligands are constitutively shed into the intercellular space and demonstrate that a step increase in compressive stress enhances EGF receptor (EGFR) phosphorylation with magnitude and onset kinetics closely matching those predicted by constant-rate ligand shedding in a slowly shrinking intercellular geometry. Despite the modest degree and slow nature of EGFR activation evoked by compressive stress, we find that the majority of transcriptomic responses to sustained mechanical loading require ongoing activity of this autocrine loop, indicating a dominant role for mechanotransduction through autocrine EGFR signaling in this context. A slow deformation response to a step increase in loading, accompanied by synchronous increases in ligand concentration and EGFR activation, provides one means for cells to mount a selective and context-appropriate response to a sustained change in mechanical environment.

The Chitinase-like Protein YKL-40 is Secreted by Airway Epithelial Cells at Base Line and in Response to Compressive Mechanical Stress

The Journal of Biological Chemistry. Sep, 2010  |  Pubmed ID: 20650887

The chitinase-like protein YKL-40, encoded by the CHI3L1 gene, is a biomarker and functional effector of chronic inflammatory and allergic diseases. In the lung it is associated with asthma severity and reduced lung function. The cellular sources of YKL-40 in human airways and the mechanisms regulating YKL-40 expression are poorly understood. We previously showed that mechanical stress similar to that experienced during bronchoconstriction triggers epithelial cell signaling through epidermal growth factor receptor (EGFR), fibrotic mediator release, and goblet cell hyperplasia consistent with airway remodeling in asthma. We now show that well differentiated normal human bronchial epithelial cells express CHI3L1 and secrete YKL-40 under base-line culture conditions. Mechanical stress (30-cm H(2)O transcellular compressive stress) applied for 3 h induces CHI3L1 expression by ∼4-fold compared with time matched controls, resulting in increased secretion of YKL-40 by 3.6-fold 24 h after onset of the 3-h stimulus. Inhibition of EGFR or MEK1/2 (ERK kinase) significantly but incompletely attenuates mechanical stress-induced up-regulation of CHI3L1 expression in normal human bronchial epithelial cells. Direct activation of EGFR utilizing EGF-family ligands induces CHI3L1 expression. Our results reveal that human airway epithelial cells are a source of YKL-40 and demonstrate that mechanical stress potently induces CHI3L1 expression leading to increased secretion of YKL-40 protein in an EGFR and MEK1/2-dependent pathway. In the asthmatic airway mechanical stress may contribute to enhanced YKL-40 levels.

Feedback Amplification of Fibrosis Through Matrix Stiffening and COX-2 Suppression

The Journal of Cell Biology. Aug, 2010  |  Pubmed ID: 20733059

Tissue stiffening is a hallmark of fibrotic disorders but has traditionally been regarded as an outcome of fibrosis, not a contributing factor to pathogenesis. In this study, we show that fibrosis induced by bleomycin injury in the murine lung locally increases median tissue stiffness sixfold relative to normal lung parenchyma. Across this pathophysiological stiffness range, cultured lung fibroblasts transition from a surprisingly quiescent state to progressive increases in proliferation and matrix synthesis, accompanied by coordinated decreases in matrix proteolytic gene expression. Increasing matrix stiffness strongly suppresses fibroblast expression of COX-2 (cyclooxygenase-2) and synthesis of prostaglandin E(2) (PGE(2)), an autocrine inhibitor of fibrogenesis. Exogenous PGE(2) or an agonist of the prostanoid EP2 receptor completely counteracts the proliferative and matrix synthetic effects caused by increased stiffness. Together, these results demonstrate a dominant role for normal tissue compliance, acting in part through autocrine PGE(2), in maintaining fibroblast quiescence and reveal a feedback relationship between matrix stiffening, COX-2 suppression, and fibroblast activation that promotes and amplifies progressive fibrosis.

Matrix Rigidity Regulates Cancer Cell Growth and Cellular Phenotype

PloS One. 2010  |  Pubmed ID: 20886123

The mechanical properties of the extracellular matrix have an important role in cell growth and differentiation. However, it is unclear as to what extent cancer cells respond to changes in the mechanical properties (rigidity/stiffness) of the microenvironment and how this response varies among cancer cell lines.

A 3-D Model of Ligand Transport in a Deforming Extracellular Space

Biophysical Journal. Dec, 2010  |  Pubmed ID: 21112275

Cells communicate through shed or secreted ligands that traffic through the interstitium. Force-induced changes in interstitial geometry can initiate mechanotransduction responses through changes in local ligand concentrations. To gain insight into the temporal and spatial evolution of such mechanotransduction responses, we developed a 3-D computational model that couples geometric changes observed in the lateral intercellular space (LIS) of mechanically loaded airway epithelial cells to the diffusion-convection equations that govern ligand transport. By solving the 3-D fluid field under changing boundary geometries, and then coupling the fluid velocities to the ligand transport equations, we calculated the temporal changes in the 3-D ligand concentration field. Our results illustrate the steady-state heterogeneities in ligand distribution that arise from local variations in interstitial geometry, and demonstrate that highly localized changes in ligand concentration can be induced by mechanical loading, depending on both local deformations and ligand convection effects. The occurrence of inhomogeneities at steady state and in response to mechanical loading suggest that local variations in ligand concentration may have important effects on cell-to-cell variations in basal signaling state and localized mechanotransduction responses.

TNF-α-converting Enzyme/a Disintegrin and Metalloprotease-17 Mediates Mechanotransduction in Murine Tracheal Epithelial Cells

American Journal of Respiratory Cell and Molecular Biology. Aug, 2011  |  Pubmed ID: 21097655

Bronchoconstriction applies compressive stress to airway epithelial cells. We show that the application of compressive stress to cultured murine tracheal epithelial cells elicits the increased phosphorylation of extracellular signal-regulated kinase (ERK) and Akt through an epidermal growth factor receptor (EGFR)-dependent process, consistent with previous observations of the bronchoconstriction-induced activation of EGFR in both human and murine airways. Mechanotransduction requires metalloprotease activity, indicating a pivotal role for proteolytic EGF-family ligand shedding. However, cells derived from mice with targeted deletions of the EGFR ligands Tgfα and Hb-egf showed only modest decreases in responses, even when combined with neutralizing antibodies to the EGFR ligands epiregulin and amphiregulin, suggesting redundant or compensatory roles for individual EGF family members in mechanotransduction. In contrast, cells harvested from mice with a conditional deletion of the gene encoding the TNF-α-converting enzyme (TACE/ADAM17), a sheddase for multiple EGF-family proligands, displayed a near-complete attenuation of ERK and Akt phosphorylation responses and compressive stress-induced gene regulation. Our data provide strong evidence that TACE plays a critical central role in the transduction of compressive stress.

Lysophosphatidic Acid Stimulates Epidermal Growth Factor-family Ectodomain Shedding and Paracrine Signaling from Human Lung Fibroblasts

Wound Repair and Regeneration : Official Publication of the Wound Healing Society [and] the European Tissue Repair Society. Mar-Apr, 2011  |  Pubmed ID: 21362091

Lysophospatidic acid (LPA) is a bioactive lipid mediator implicated in tissue repair and wound healing. It mediates diverse functional effects in fibroblasts, including proliferation, migration and contraction, but less is known about its ability to evoke paracrine signaling to other cell types involved in wound healing. We hypothesized that human pulmonary fibroblasts stimulated by LPA would exhibit ectodomain shedding of epidermal growth factor receptor (EGFR) ligands that signal to lung epithelial cells. To test this hypothesis, we used alkaline phosphatase-tagged EGFR ligand plasmids transfected into lung fibroblasts, and enzyme-linked immunosorbent assays to detect shedding of native ligands. LPA induced shedding of alkaline phosphatase-tagged heparin-binding epidermal growth factor (HB-EGF), amphiregulin, and transforming growth factor-a; non-transfected fibroblasts shed amphiregulin and HBEGF under baseline conditions, and increased shedding of HB-EGF in response to LPA. Treatment of fibroblasts with LPA resulted in elevated phosphorylation of extracellular signal-regulated kinase 1/2, enhanced expression of mRNA for c-fos, HB-EGF and amphiregulin, and enhanced proliferation at 96 hours. However, none of these fibroblast responses to LPA required ectodomain shedding or EGFR activity. To test the ability of LPA to stimulate paracrine signaling from fibroblasts, we transferred conditioned medium from LPA-stimulated cells, and found enhanced EGFR and extracellular signal-regulated kinase 1/2 phosphorylation in reporter A549 cells in excess of what could be accounted for by transferred LPA alone. These data show that LPA mediates EGF-family ectodomain shedding, resulting in enhanced paracrine signaling from lung fibroblasts to epithelial cells.

Physical Forces and Airway Remodeling in Asthma

The New England Journal of Medicine. May, 2011  |  Pubmed ID: 21612476

A Multiwell Platform for Studying Stiffness-dependent Cell Biology

PloS One. 2011  |  Pubmed ID: 21637769

Adherent cells are typically cultured on rigid substrates that are orders of magnitude stiffer than their tissue of origin. Here, we describe a method to rapidly fabricate 96 and 384 well platforms for routine screening of cells in tissue-relevant stiffness contexts. Briefly, polyacrylamide (PA) hydrogels are cast in glass-bottom plates, functionalized with collagen, and sterilized for cell culture. The Young's modulus of each substrate can be specified from 0.3 to 55 kPa, with collagen surface density held constant over the stiffness range. Using automated fluorescence microscopy, we captured the morphological variations of 7 cell types cultured across a physiological range of stiffness within a 384 well plate. We performed assays of cell number, proliferation, and apoptosis in 96 wells and resolved distinct profiles of cell growth as a function of stiffness among primary and immortalized cell lines. We found that the stiffness-dependent growth of normal human lung fibroblasts is largely invariant with collagen density, and that differences in their accumulation are amplified by increasing serum concentration. Further, we performed a screen of 18 bioactive small molecules and identified compounds with enhanced or reduced effects on soft versus rigid substrates, including blebbistatin, which abolished the suppression of lung fibroblast growth at 1 kPa. The ability to deploy PA gels in multiwell plates for high throughput analysis of cells in tissue-relevant environments opens new opportunities for the discovery of cellular responses that operate in specific stiffness regimes.