Our study demonstrates that MTECs in ALI cultures respond to compressive stress in a manner similar to, but not identical with, human airway epithelial cells cultured under similar conditions. During bronchoconstriction, as may occur in asthma, the constriction of smooth muscle generates a compressive force on the order of 30 cm H2
O on airway epithelial cells (28
). We found that a transepithelial cell layer pressure gradient of 30 cm H2
O induced a peak response in the phosphorylation of ERK1/2 through EGFR signaling in MTECs. This finding is similar to our previous findings in NHBE cells (5
). We also found that mechanical stress up-regulated the gene expression of EGF-family ligands in MTECs, with a peak at 4 hours.
Some differences were evident in the response of MTECs and NHBE cells. In MTECs, amphiregulin was most highly up-regulated by compressive stress, whereas in NHBE cells, HB-EGF was most highly up-regulated. However, both MTECs and NHBE cells exhibited the activation of the same EGFR-dependent pathways in response to compressive stress. This similarity ( and ) provided a basis for using this model to elucidate how mechanical stress is linked to the biological activation of airway epithelial cells, using cells from genetically manipulated mice.
We first examined mice with a deletion of Hb-egf
, based on previous experiments in human cells where a neutralizing antibody to HB-EGF appeared effective at attenuating mechanotransduction (7
). Although we expected that cells derived from these animals would not respond to compressive stress, we found that the signal transduction response was not distinguishable from that observed in cells derived from littermate control mice without the deletion. We considered that species differences might exist between humans and mice. In fact, Hb-egf
was not up-regulated in response to mechanical stress in MTECs. We also speculate that mice born without Hb-egf
may develop compensatory pathways for signaling that replace Hb-egf
Based on evidence that TGF-α plays a critical role in autocrine EGFR phosphorylation in airway epithelial cell lines (20
) and that human bronchial epithelial cells shed TGF-α (9
), we explored mechanotransduction responses in cells harvested from mice with a targeted deletion of Tgfα
. We found that the ERK1/2 phosphorylation response to compressive stress was partially attenuated in these cells (~ 36% reduction). This attenuation was not further modified by the addition of neutralizing antibodies against the EGF-family ligands epiregulin and amphiregulin.
Our study was limited insofar as blocking all EGF-family ligands is impossible, because of the lack of availability of neutralizing antibodies for all murine EGF family members. Taken together, our data allow us to conclude that TGF-α plays a contributing role in the phosphorylation of ERK1/2 induced by compressive stress in MTECs, but the genetic deletion of TGF-α alone only partly attenuates the mechanotransduction response.
However, we know that other (likely multiple) EGFR ligands could also contribute to mechanotransduction responses, based on the abundant expression of amphiregulin, epiregulin, and betacellulin. EGF is unlikely to play as important a role, because the EGF mRNA in MTECs was minimally detectable compared with that for TGFα, amphiregulin, epiregulin, HB-EGF, and betacellulin, and the expression of EGF mRNA was not up-regulated by mechanical stress. We attempted to measure the direct release of EGFR ligands in our system, but were unsuccessful. This failure likely occurred because after EGFR ligands are released, the soluble ligands are captured by EGFR, making detection in the cell supernatant difficult (29
). In our previous study of NHBE cells, to detect EGFR ligands (EGF and TGF-α) in the cell-culture medium, we needed to add a neutralizing EGFR antibody (9
). Because no mouse-specific neutralizing EGFR antibody was available, we could not detect any EGFR ligands in our cell supernatants.
Although we were unable to pinpoint a specific EGFR ligand responsible for linking mechanical compression to biological signal transduction, all EGFR ligands are released from cell membranes by sheddases (13
), that is, enzymes that cleave membrane-bound precursors of a ligand, leading to signal transduction in the microenvironment of its release. We reasoned that an individual sheddase could impose a rate-limiting step in the mechanotransduction cascade. Our initial data were consistent with a pivotal role for shedding in mechanotransduction, in that the inhibition of metalloproeases with the small molecule inhibitors GM6001 and TAPI-2 abrogated the ERK1/2 and Akt phosphorylation responses to mechanical stress (). These findings were in agreement with results from NHBE cells (7
). However, because these inhibitors are not specific for any single metalloprotease or class of metalloproteases, our results did not allow us to determine which specific sheddase links mechanical stimulation to downstream biological events.
Among known sheddases, tumor necrosis factor-α–converting enzyme (TACE/ADAM17), we hypothesized, was the major enzyme responsible for the cleavage of EGFR ligand precursors from the cell membrane. TACE is a member of the ADAM family, a group of zinc-dependent transmembrane metalloproteases (16
) and a major sheddase for TGF-α, amphiregulin, epiregulin, and HB-EGF (11
knockout mice are perinatal-lethal, and their phenotype resembles that of mice with a targeted deletion of EGFR
, sharing features with Tgfα
–deficient, or amphiregulin
-deficient mice (11
). The overlapping phenotypes of mice lacking these growth factors and those lacking TACE support a critical role for the soluble forms of EGFR ligands, and imply that the proteolytic release of EGFR ligands is fundamental in EGFR signaling.
To test the role of TACE in mechanotransduction, we bred floxed Tace
) with mice expressing a tamoxifen-inducible Cre recombinase (34
). We induced Cre activity after cells were isolated and in culture, based on the knowledge that the activation of autocrine EGFR plays a key role in airway epithelial proliferation. Thus we anticipated that cells harvested from mice in which Tace
was already deleted by the activation of Cre in vivo
might not be viable for our cell-culture experiments.
In control experiments, we found that incubating MTECs derived from normal mice in ALI culture with 1 μM 4-OH tamoxifen for 14 days (35
) had no impact on responses to compressive stress. Therefore, the duration of tamoxifen exposure needed for the removal of floxed Tace
alleles would not affect our results. Cells derived from mice with the floxed Tace
allele, when activated by tamoxifen, exhibited markedly decreased (> 80%) responses to compressive stress (). In contrast, cells from the same mice cultured in the absence of tamoxifen transduced compressive stress in a normal fashion. These results demonstrate that TACE is the dominant sheddase necessary for the transduction of compressive stress through the activation of EGFR. Our data do not allow us to distinguish between an increased activation of TACE by compressive stress or simply greater access of already activated TACE to membrane-bound precursor ligands. Further research will be needed to determine the specific mechanisms at work.
The modest residual response of tamoxifen-treated MTECs derived from Taceflox/flox R26Cre+ ER
mice could involve the manifestations of at least two possible mechanisms, residual TACE protein activity or the contributions of an alternative sheddase. Extending tamoxifen exposure to 19 days did not resolve the residual mechanotransduction response, arguing against a measurable contribution from residual TACE protein, which should diminish with prolonged tamoxifen exposure. In a murine model of asthma, both ADAM10 and TACE were overexpressed in the lungs (37
). Because ADAM10 can shed EGFR ligands, including TGF-α (38
) and HB-EGF (39
), ADAM10 could be responsible for the residual response to compressive stress. Consistent with this explanation, recent studies demonstrated that ADAM10 plays a role in the TGF-α processing of Tace
-deficient cells (40
), and ADAM10 is a major sheddase for betacellulin (31
), which was one of the EGF-family ligands up-regulated by compressive stress in MTECs.
Recent evidence suggests that a variety of stimuli can trigger the activation of EGFR in airway epithelium, and that EGFR plays important roles in airway biology. Thus the demonstration of a dominant role for TACE as a sheddase in airway epithelium has potentially broad implications. Several lines of evidence link the activation of EGFR and asthma, and in particular, the remodeling of airway epithelium in animals (42
) and human cells (43
). We reported that EGFR-dependent mechanosignaling contributes to mucous cell hyperplasia in response to repeated bouts of compressive stress (45
). TACE was previously linked to the regulation of mucin gene expression in cultured human epithelial cells (20
), although the link was only definitively documented in the NCI-H292 cell line. Another study demonstrated that TACE mediates cell proliferation via the shedding of amphiregulin in response to cigarette smoke, again in NCI-H292 cells (46
). Our study (to the best of our knowledge, for the first time) definitively identified a dominant role for the TACE-dependent activation of EGFR in primary airway epithelial cells grown under well-differentiated conditions in ALI cultures.
Our data extend previous findings by demonstrating that compressive stress also initiates the phosphorylation of Akt in a pressure-dependent manner (). Studies of the ovalbumin-induced allergic asthma murine model showed that the serine phosphorylation of Akt is part of the “asthma-like” response observed in these mice (47
). The finding that mechanical stress can provoke this asthma-related signal echoes previous observations that mechanical stress responses in airway epithelial cells mimic many of the molecular events characteristic of asthmatic airway remodeling (2
). Although redundant EGFR ligands appear to occur in the signaling cascade, the cleavage of these ligands is largely restricted to TACE, making it a potential target for limiting airway remodeling, as is known to occur in patients with asthma. (). Further work will be needed to determine if TACE activity is regulated biochemically or by variations in the access of EGFR ligands to TACE.
Figure 6. Proposed mechanism of mechanical stress–induced activation of EGFR pathway in murine tracheal epithelial cells. The compressive stress–induced activation of EGFR depends on the TACE-dependent shedding of EGF-family ligands. The activation (more ...)
In conclusion, we demonstrate in MTECs that the compressive stress–induced phosphorylation of ERK1/2 and Akt through EGFR signaling is a metalloprotease-dependent process (). Our data indicate a dominant role for TACE as the key sheddase involved in mechanically driven signal transduction in MTECs. These findings provide new insights into the mechanisms regulating mechanotransduction in MTECs.