The data presented here support the hypothesis that oxidative stress–induced GC metaplasia of differentiated primary cultures of human airway epithelial cells is due, at least in part, to a cascade of events initiated by the breakdown of HA, the activation of TK, the release of mature EGF, and EGFR activation. We have previously shown that HA is present at the apical pole of airway epithelial cells (47
) and that it is bound to TK, inhibiting its catalytic activity (41
); therefore, we expected that ROS-induced HA depolymerization resulted in TK activation. We did not test if, in addition to the TK release from HA–TK complexes, X/XO induced increased TK expression. If this occurs, HA depolymerization would be necessary for the release of active TK because high-molecular-weight HA present at the epithelial surface would be sufficient to control the catalytic activity of the increased levels of TK. We also anticipated that TK should be able to process pro-EGF as reported in primary cultures of human submucosal glands cells and mimic ROS effects (39
). The novel findings in this study were that HA depolymerization induced by ROS resulted in phenotypic changes on differentiated primary cultures of NHBE cells using a relatively short-term exposure (once daily for 3 d) to oxidative stress. This treatment led to GC metaplasia, which was associated with EGFR activation by EGF. In the present study, we did not address the origin of the increased GC population, but the metaplasia is likely the result of proliferation from basal cells, as has been reported (63
), or differentiation from nonproliferating pre-existent cells in combination with the increased half life due to upregulation of antiapoptotic factors (22
Our data confirm findings by other groups who reported that EGFR signaling was shown to result in MUC5AC induction, although there are with some discrepancies regarding to the ligands responsible for EGFR signaling. We found that in NHBE cells grown at the ALI, the ligand responsible for EGFR activation under oxidative stress was EGF, processed from its transmembrane pro-form by TK. The inhibitory effect of neutralizing anti-EGF antibodies on ROS-induced EGFR and MAPK activation confirmed these findings.
In contrast, other reports have shown that oxidative stress–induced GC metaplasia or MUC5AC expression was associated with TGF-α (66
), amphiregulin (68
), or HB-EGF (13
). The activation of all the transmembrane pro-ligands mentioned previously requires the activity of metalloproteases of the ADAM family. In our system, metalloproteases seem not to play a role in GC metaplasia because responses were not inhibited by the metalloprotease inhibitor GM6001. Confirming this notion, we found that EGF and not TGF-α levels increased in response to oxidative stress in our cells; these results are in contrast to the report of Shao and colleagues (67
), who reported that H2
production induced by PMA resulted in an increase of TGF-α−mediated EGFR activation. There are several explainations for the discrepancies between the studies. First, Shao and colleagues used a much lower concentration of retinoic acid in their culture media (~ 3 × 10−10
M versus 5 × 10−8
M in our cultures). This is likely relevant because TGF-α gene expression is induced by these low concentrations of retinoic acid (70
). We used the concentration that Koo and colleagues (71
) found to be necessary to maintain mucous cell differentiation in NHBE cell cultures. Second, primary cells cultures, unlike cell lines, contain a heterogeneous cellular population (e.g., ciliated, secretory, basal) and show greater variability in a number of responses (72
). Our studies were performed in Passage 1 cells with > 80% ciliated cells. Under these conditions, our initial number of GCs was ~ 3%. Third, the initial culture conditions, such as the characteristics of the cultures (number of ciliated and goblet cells), the cell passage, and the media composition used by Shao and colleagues (67
), were not fully detailed, and therefore a comparison with our initial conditions is not possible. Fourth, differences such as in the stimuli used to induce ROS production (PMA instead of X/XO) may be implied in activating different enzymes (TACE versus TK). Last, EGF was removed from our culture media 48 h before and during treatments (to allow us to assess the role of the endogenous, membrane-bound pro-EGF processing). The fact that those authors have used media supplemented with EGF may have affected the endogenous synthesis of pro-EGF.
Other stimuli, such as IL-13 or neutrophil elastase (73
), have been shown to induce GC metaplasia (74
) and epithelial cell proliferation (77
) by metalloprotease activation (e.g., TACE) and TGF-α release on bronchial epithelial cells (76
) or by enhancing MUC5AC RNA stability (73
). The pathways involved in GC metaplasia and hypersecretion using those stimuli differ but do not conflict with our observations. For instance, primary bronchial or nasal epithelial cells were treated with IL-13 from the moment that they were exposed to air. Under these conditions, differentiation into GC phenotype was higher than in epithelial cells grown in the absence of IL-13 (74
). In contrast to our ROS exposure of completely differentiated NHBE cells (~ 21 d on air and with > 80% of ciliated cells) once daily for 3 d, the exposure to IL-13 was lengthy (14 d) starting as soon as cells were exposed to air (~ 7 d) and during the differentiation stage. Therefore, differences in experimental conditions and stimuli used in those reports likely explain the discrepancies in the EGFR ligands involved in GC metaplasia.
In our cultures, the basolateral distribution EGFR is modified by oxidative stress, which induced EGFR expression and/or translocation to the apical surface of NHBE cells. This change in localization allows ligand/receptor interaction because EGF (and most ErbB receptor ligands) is expressed at the apical compartment of epithelial cells and is separated by tight junctions from its receptor (78
), which is localized basolaterally in normal epithelium (29
). This pattern has been described in the airway epithelium of smokers (79
) and patients with asthma (80
), suggesting that oxidative stress could mediate EGFR apical expression and/or translocation. This study also shows that EGFR is colocalized with MUC5AC in GC, as has been described in the epithelium of patients with asthma (27
), suggesting that these cultures are a useful model to assess epithelial responses to oxidative stress.
In addition, we found that X/XO induced Bcl-2 gene expression and that this effect was mimicked by rTK. In Bcl-2, protein expression was not evident by immunohistochemistry in the control cells but was highly expressed after rTK treatment. These results are in agreement with Bcl-2 increases induced by EGF and p44/42
MAPK in other tissues (81
). Thus, EGFR activation resulted in an additional mechanism aimed at sustaining increased GC number by slowing the turnover rate of these cells during oxidative stress. These effects were long lasting: Bcl-2 gene expression and the number of GC remained high after those cultures were exposed to normal ALI conditions for three additional days after the chronic treatment with X/XO. These observations seem to be clinically relevant to hypersecretory states associated with human airway diseases. For example, an increased Bcl-2 immunoreactivity has been described in the airways of patients with asthma (21
) and in mucous cells from patients with cystic fibrosis (20
). The fact that inhibition of Bcl-2 expression resulted in reduction of GC metaplasia (20
) suggests that a decrease in GC turnover could contribute to the observed increase in GC population in human epithelium in such conditions.
We found that another GC product, LPO (23
), was induced by rTK treatment. This is consistent with the notion that GC, in addition to increased mucins in response to oxidative stress, are also capable of increasing the ROS scavenging properties of airway mucus through LPO (23
In summary, these results provide a direct mechanistic link between sustained airway ROS insult during inflammatory responses and the development of GC metaplasia and mucous hypersecretion, characteristics of asthma and COPD. Our results suggest that HA plays a key role in regulating oxidative stress–induced GC metaplasia in human airways by regulating TK activity. The role of specific oxidants from exogenous (e.g., tobacco smoke) or endogenous [i.e., H2
generation mediated by Duox (84
)] sources in HA depolymerization and EGFR-mediated GC metaplasia needs further investigation.