Division of Therapeutics and Molecular Medicine, University of Nottingham, Nottingham NG7 2UH, United Kingdom
Submitted 3 July 2003 ; accepted in final form 8 November 2003
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ABSTRACT |
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BEAS-2B; apoptosis; basement membrane; insulin; integrins
Whereas epithelial cells can be shed from the epithelium by direct mechanical damage (e.g., at bronchoscopy), a second potential mechanism for the disruption of the epithelium is cell death. Epithelial cell death may be brought around by necrosis, usually secondary to inhalation of toxins, or potentially by apoptosis. Epithelial cells from many epithelial surfaces will die through apoptosis if removed from their cell-matrix attachments, a phenomenon first observed in mammary epithelium, termed "anoikis" (6). A similar mechanism of apoptosis induction is also apparent in the bronchial epithelium (2). However, the background rate of apoptosis in intact epithelia in vivo appears to be very low (24), suggesting that in this setting, epithelial cells receive a strong survival signal acting to prevent the onset of apoptosis. The aim of the current study was to extend earlier observations on the nature of the survival signals that airway epithelial cells receive and, in particular, to determine the role of matrix in these responses. Here, we report that a number of important mediators/cytokines can promote survival signaling in airway epithelial cells and also that a key role for matrix exists in airway epithelial cell survival signaling. Furthermore, airway epithelial cells are able to synthesize matrix that provides a key survival signal. These general mechanisms operate in both a model cell line (BEAS-2B) and in cultured primary bronchial epithelial cells.
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METHODS |
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Primary human bronchial epithelial cells (HBECs) were purchased from Clonetics and were cultured to passage 4, using bronchial epithelial growth medium (BEGM, Clonetics). BEGM consisted of bronchial epithelial basal medium (BEBM) with growth factor supplements (as used in SRM) supplied by Cambrex. All experiments were conducted at passage 4 with cells seeded in BEBM without growth factor supplements onto uncoated or ECM-coated 96-well plates. Cells were cultured for 48 h as described above.
ECM preparation. ECM-coated dishes were produced by incubating wells of a 96-well plate with 50 µl of 10 µg/ml of fibronectin (Sigma), collagen IV (Sigma), laminin I (Calbiochem), vitronectin (Calbiochem), collagen I (Sigma), collagen V (Sigma), tenascin-C (Chemicon), and elastin (Sigma) overnight. In other experiments, we coated wells with a biosynthesized matrix using a previously published method (7). Briefly, cells were seeded in serum-free SRM (BEAS-2B) or BEGM (HBECs) onto plastic, flat-bottomed 96-well plates and incubated until confluent. Cells were then removed using 0.02 M NH4OH to leave behind an intact, biosynthesized matrix (BSM). Wells were also coated with serum proteins by incubating with 10% FCS in DMEM without cells present for 48 h.
BSM protein quantification. To quantify BSM protein content, flat-bottomed 96-well plates were coated with BSM as described above; control wells were coated with fibronectin at a range of concentrations. A Bradford-based protein assay kit (Bio-Rad) was used to stain protein adsorbed to the bottom of BSM- or fibronectin-coated wells. Wells were washed with PBS, and the absorption at 595 nm of BSM-coated wells was compared with the fibronectin standards to provide a quantitative value for BSM protein content.
Apoptosis assays. Cells were seeded at a density of 0.8-1.2 x 105 cells/ml into 96-well flat-bottomed dishes (Nunc) in DMEM with serum or growth factors added as indicated and cultured for 48 h at 37°C and 5% CO2. In some experiments, the protein synthesis blocker cycloheximide (CHX; 50 µM, Sigma) was added to wells before cell seeding, as used in previous studies, to prevent de novo synthesis of ECM (5).
Integrin-matrix interaction. For relevant experiments before seeding onto matrix-coated wells, cells were kept in suspension for 15 min at 37°C with either the RGD-blocking GRGDS peptide (200 µg/ml; Calbiochem) or the inactive analog GRADS (200 µg/ml; Novabiochem).
Evaluation of apoptosis by fluorescence microscopy. Cells in 96-well plates were fixed in situ for 30 min with 4% paraformaldehyde and stained with 1.5 µg/ml of propidium iodide (PI, Sigma). Stained cells were observed by fluorescence microscopy (x200; Nikon Diaphot 300), and apoptotic cells were morphologically identified by the presence of condensed, brightly stained nuclei and reduced cytoplasm. The total number of healthy and apoptotic cells was calculated for three random fields per well, in triplicate wells in three separate plates, and the percentage of apoptotic cells was calculated. All counts were performed with the observer blinded to treatments.
Evaluation of apoptosis by DNA fragmentation. To confirm the results of the PI counting assay for apoptosis, we used a TdT-mediated dUTP nick end labeling (TUNEL)-based assay. Briefly, a TiterTACS kit (R&D Systems) was used to measure DNA fragmentation, a late sign of apoptosis. Cells were cultured as outlined earlier in a variety of apoptosis-stimulating and survival-inducing conditions. After 48 h, cells were fixed and DNA breaks in apoptotic cells were detected using a TdT enzyme linked to a horseradish peroxidase amplification system. The number of apoptotic cells in a well was proportional to the absorbance at 450 nm. We also performed cell counts by counting total cell number in three random fields per condition in triplicate wells. Over the course of the incubation period, a significant increase in cell number had occurred in healthy cultures. We therefore corrected the results for cell number by dividing the absorption value obtained from the TiterTACS assay by the total cell number counted for each experimental condition, the resulting value being proportional to the percentage of apoptotic cells present in the sample.
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RESULTS |
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Effect of serum and soluble growth factors on BEAS-2B apoptosis. Our initial experiments were designed to confirm the previously observed dependence of airway epithelial cell survival on the presence of serum in the culture medium. Figure 1 shows that BEAS-2B cells, when deprived of serum at seeding, exhibit increased levels of apoptosis up to a maximum at 48 h of 19.0 ± 1.4% (n = 3). However, when these experiments were performed in the presence of the protein synthesis inhibitor CHX (50 µM), apoptosis rates were markedly higher (81.4 ± 3.3%, n = 3, P < 0.001). The CHX-induced cell death response was inhibited (although not back to baseline levels) by the presence of serum (Fig. 1), suggesting that factors present in serum could, to a large extent, provide the necessary survival signal required to prevent the onset of apoptosis.
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We next examined the survival effects of a range of mediators and cytokines to which airway epithelial cells are likely to be exposed in vivo. PDGF, transforming growth factor-, serotonin, histamine, and bradykinin all failed to significantly prevent serum deprivation-induced cell death (data not shown). In contrast, IGF-I, IGF-II, and insulin all provided strong concentration-dependent survival signals (Fig. 2, A-C). The survival signals from EGF and FGF were less potent but still significant (Fig. 2, D and E).
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The logEC50 ± SE values for inhibition of apoptosis for these agents were: IGF-I = -7.28 ± 0.15, insulin = -6.42 ± 0.31, IGF-II = -7.34 ± 0.28, EGF = -9.56 ± 0.45, and FGF = -8.96 ± 0.40 (all units log g/ml, n = 3).
To verify the survival effects observed using our PI staining counting method, we used a TiterTACS (R&D systems) TUNEL-based assay to detect DNA breaks in cells undergoing late-stage apoptosis. Results were corrected for cell number and expressed as a % of serum withdrawal (Fig. 3). A similar pattern of results was observed using both PI counting and TiterTACS methods.
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Together, these results demonstrate that a number of survival factors inhibits apoptosis induced by serum withdrawal. However, despite the above effects, we found these soluble growth factors were unable to reverse CHX-induced cell death as effectively as serum (data not shown). We also observed that confluent monolayers treated with CHX did not undergo a significant increase in apoptosis (Fig. 4), complementing previous studies in a mammary epithelial cell line that show CHX treatment does not influence anoikis levels of detached cells (26). Because an intact monolayer will already possess an ECM substrate, we hypothesized that an ECM component of serum may be responsible for the greater protective effect observed with serum exposure. Therefore, we next studied the effect of matrix present in serum and of matrix secreted by cells on survival in the presence of CHX.
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Effect of insoluble factors on BEAS-2B apoptosis. First, we observed that coating wells with serum protein provided similar levels of protection to those seen with the addition of serum into culture medium, with CHX-induced cell death being inhibited by 69.0 ± 4.7% (Fig. 5). However, pretreatment with SRM, which contains a variety of growth factors (see METHODS), failed to replicate this survival effect, suggesting that a component of serum not present in SRM was responsible. We also found that chemically stripping confluent cells from culture plates left behind a BSM secreted by these cells attached to the bottom of the wells. This BSM replicated the survival effect of serum when cells were replated onto it (all n = 3, Fig. 5). Interestingly, the amount of matrix secreted by BEAS-2B cells was high after 48 h in culture, being 25 ± 0.4 µg/ml (equivalent to a protein concentration of 3.91 ± 0.06 µg/cm2; n = 5). Together, these data suggest that airway epithelial cells in culture are able to rapidly modify their extracellular environment by secreting matrix factors that provide a key survival signal for the cells.
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Effects of individual matrix factors on BEAS-2B apoptosis. To define the key matrix factors that provide the survival signal for BEAS-2B cells, we used the model of protection from CHX-induced apoptosis described above. We precoated wells with single matrix factors at concentrations (10 µg/ml) similar to that of biosynthesized matrix we had previously observed to be secreted and examined apoptosis rates when cells were seeded in the presence of CHX (Fig. 6A). Under these conditions, apoptosis rates on uncoated plastic wells were high (95.5 ± 1.1%). A significant survival effect was observed with fibronectin (54.5 ± 3.3%), vitronectin (48.3 ± 2.8%), collagen I (32.9 ± 3.3%), collagen IV (53.7 ± 4.4%), BSM (54.5 ± 3.3%), and laminin (31.3 ± 2.8%), which all provided rescue from apoptosis (n = 3). Elastin, tenascin-C, and collagen V did not confer any protection (all n = 3). The pattern of survival effects of matrix was similar when serum deprivation (in the absence of CHX) was used as the apoptotic stimulus (Fig. 6B), although as shown in Fig. 1, the overall rates of apoptosis were lower than experiments in which CHX was included. In these experiments, collagen V and elastin increased apoptosis levels by 74.6 ± 6.6% and 40.0 ± 7.7%, respectively (n = 3).
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Effect of RGD-blocking peptide on ECM rescue from apoptosis. Airway epithelial cells such as the BEAS-2B cell line are known to express a range of integrin receptors (25). Based on the pattern of survival effects observed with matrix factors, we hypothesized that the strongest candidate interaction for the survival signal seen in these experiments was interaction of RGD motifs present on matrix with cell surface integrins. We therefore studied the effect of the RGD blocking, short peptide antagonist GRGDS, and a relevant inactive control peptide (GRADS) on rescue from CHX-induced cell death provided by matrix factors. In keeping with a role of interaction with RGD-binding integrins, GRGDS peptide inhibited survival signaling for several of the matrix factors studied (Fig. 7, A and B). GRGDS inhibition of ECM-mediated survival reached statistically significant levels on fibronectin (84.9 ± 4.0% inhibition of rescue, n = 5, P < 0.001), vitronectin (95.5 ± 2.9%, n = 3, P < 0.001), collagen I (92.3 ± 5.3%, n = 6, P < 0.001), and serum protein (81.0 ± 4.4%, n = 5, P < 0.001). Rescue by laminin (n = 3, P > 0.05), BSM (n = 6, P > 0.05), and collagen IV (n = 6, P > 0.05) was inhibited slightly by GRGDS preincubation, but not to a statistically significant level (Fig. 7, A and B). In this series of experiments, cell death was presumably occurring via anoikis as cell-ECM interactions were being blocked by the GRGDS peptide; however, loss of detached cells into the supernatant was low and the majority of apoptotic cells was still present on the well bottom.
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Effect of soluble growth factors on primary HBEC apoptosis. Although BEAS-2B cells are a good model system for the study of airway epithelial responses, it is conceivable that survival signaling may be altered in this cell line from that present in a nontransformed cell system. Therefore, we repeated the key experiments from the series described above in a primary airway epithelial cell system using undifferentiated HBECs. The first observation we made was that levels of apoptosis observed in HBECs on growth factor withdrawal were significantly higher than in the BEAS-2B system (96.2 ± 1.04% in HBECs cf. 19.0 ± 1.4% in BEAS-2B, n = 3). Experiments involving the addition of individual soluble growth factors confirmed that like BEAS-2B cells, HBECs also receive a survival signal from the insulin family of growth factors, although the level of protection is weaker than in the cell line. Insulin, IGF-I, and IGF-II (Fig. 8, A-C, respectively) all showed concentration-dependent protective effects. EGF (Fig. 8D) demonstrated a weaker survival effect. FGF did not show any effect on cell survival in our experiments (data not shown). Interestingly, adhesion experiments showed insulin did not affect the adherence of HBECs to uncoated plastic wells at an early time point (5 h postseeding), thus suggesting insulin promotes HBEC survival without promoting cell-substratum adherence (data not shown).
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Effect of ECM on HBEC apoptosis. The overall pattern of ECM-mediated rescue observed in the HBEC system was similar to that in the BEAS-2B cell system (Fig. 9). As before, growth factor withdrawal induced a higher level of apoptosis in the HBECs (67.9 ± 4.8% in this set of experiments) compared with the transformed cell line. Collagen IV (78.7 ± 5.9%) and BSM (74.4 ± 5.2%) conferred the highest level of rescue. Fibronectin (42.1 ± 6.5%), laminin (51.3 ± 6.0%), and collagen I (40.7 ± 7.1%) showed statistically significant but less effective protection (n 3, P < 0.01). Tenascin-C and elastin did not show statistically significant levels of protection, whereas collagen V induced an increase in HBEC apoptosis to above control levels (43.1 ± 4.8% increase, n = 4, P < 0.01). Although vitronectin provided a survival signal for BEAS-2B cells, we did not observe any protection by this matrix factor in the primary HBEC system.
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DISCUSSION |
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We found primary HBECs were in general more prone to undergo apoptosis than cells of the BEAS-2B cell line, perhaps unsurprising considering the potentially unlimited subculture capacity of the cell line compared with the very limited lifespan of the primary cells in vitro. The insulin family of growth factors brought about an increased survival rate in both the primary cells and transformed cell line without having any effect on early stage adhesion to the substratum. Levels of protection were lower in primary cells, again reflecting their greater tendency to undergo apoptosis. We also observed profound survival effects for a number of matrix factors, including fibronectin, collagens I and IV, laminin, and vitronectin. Cell survival resulting from the integration of signals from matrix-binding integrins and signaling from the insulin receptor are in keeping with previous observations in other epithelial cell systems (4, 13).
These matrix responses are highly likely to be mediated through interaction between cell surface integrins and matrix motifs. One of the most common integrin-binding matrix motifs is the RGD sequence. Many integrins bind to this motif, including the vitronectin receptor v
3, the vitronectin/fibronectin receptor
v
5, the fibronectin/tenascin-C receptor
v
6, and the fibronectin receptor
5
1. The laminin/collagen integrin receptors
2
1 and
3
1 show only weak RGD interactions (21). Our data suggest a key role for the RGD motif present on a number of key matrix factors such as fibronectin, vitronectin, and collagen I. The RGD motif does not seem to play a crucial role in adhesion to laminin, collagen IV, or biosynthesized matrix. This contrasts with previous work demonstrating survival signaling by BSM is RGD dependent (2). In our experiments, we did observe a small inhibition of BSM-mediated rescue with the RGD-blocking peptide, but not to statistically significant levels. As it is likely that BSM consists of several different matrix factors signaling through different recognition sequences, it is possible a small proportion of the BSM survival signal may be via an RGD-dependent factor such as fibronectin or vitronectin. However, the lack of a major RGD-dependent survival signal on BSM suggests the matrix secreted by BEAS-2B cells consists mainly of the normal basement membrane proteins, laminin, and/or collagen IV. This supports previous work demonstrating the ability of HBECs to secrete and adhere to laminin-5 in culture (14).
The pattern of matrix-mediated cell survival was similar in the two cell types we tested but not identical. Vitronectin provided a strong survival signal in the BEAS-2B cells but had no effect in the primary HBEC system. A more subtle difference was the greater protective effect of collagen IV compared with other ECM factors in HBECs. Previous work has shown differences in integrin expression profile between cultured HBECs and the BEAS-2B cell line, both of which differ from normal bronchial epithelium in vivo (17, 25). These data could provide a likely explanation for the different ECM effects observed in the primary cells and the cell line. The absence of vitronectin-mediated rescue in cultured HBECs is probably due to the lack of expression of the v
3-integrin. The
v
3-integrin acts as a vitronectin receptor, is expressed in the BEAS-2B cell line (25), and most likely mediates protection from apoptosis in BEAS-2B cells, as previously observed in an
v
3-transfected HEK-293 cell line (3). The strong survival effect of collagen IV in HBECs may also be due to a difference in integrin expression as the laminin/collagen IV-binding
6
4-integrin is expressed by HBECs in vitro but not by BEAS-2B cells (25). Although this receptor predominantly interacts with laminin, the strong survival effect of collagen IV observed in HBECs may be due to the presence of this integrin. The observation in the primary and transformed cell systems that collagen V causes a significant increase in apoptosis demonstrates that, depending on the ligand engaged, matrix signaling can be both pro- and antiapoptotic.
The previous variations observed in integrin expression profile between primary HBECs and the BEAS-2B cell line, in conjunction with the differences we observed in matrix rescue, suggest that conclusions drawn from matrix studies using transformed epithelial cell lines should be made carefully since responses may be different than those seen with primary cells. Because there is also some variation in integrin expression between the epithelium in vivo and primary cells in culture [for example, the tenascin-C receptor 9
1-integrin is present in vivo but not in vitro (25)], caution also needs to be exercised when relating specific matrix effects observed in the primary systems in vitro to the intact bronchial epithelium.
In vivo, cells at the basolateral surface of the airway are in close contact with the basement membrane. Immunohistochemical studies have shown little matrix to be present in the airway epithelium above the level of the true basement membrane, but epithelial cells maintain cell-cell contact via tight junctions, adherens junctions, and desmosomes (20). Therefore, it seems likely that the integrity of the airway epithelium depends first on interaction of cells in the basal layer of the epithelium with the basement membrane and second on maintenance of cell-cell contact. The relative proportion of matrix factors changes during airway inflammation. For example, tenascin-C, which is normally absent from the airway epithelium, is present in the basement membrane of asthmatics, with expression levels correlated to disease severity (11). These matrix changes may lead to an alteration in the ability of the immediate cell environment to provide the requisite survival signals. Laminin isoforms are also altered in the asthmatic basement membrane, possibly downregulating the survival signals received by the basal cells of the epithelium, increasing apoptosis and cell loss (1). Conversely, asthmatic matrix changes may also have a protective effect. A characteristic feature of asthma is thickening of the reticular basement membrane due to increased collagen deposition by myofibroblasts (19). This may increase the load on the underlying smooth muscle cells, providing an increased resistance to airway narrowing (15).
In addition to survival effects, one major consequence of growing epithelial cells upon differing matrix factors is a change in cell phenotype, which in turn alters the ability of cells to maintain their cell-cell contacts and can influence migration (22, 23). In vivo, the situation is complicated by changes in not just one but a number of matrix factors and, in addition, our data clearly show that there is redundancy in the ability of matrix factors to provide survival signals. Although our studies point to an important role for the interaction of matrix factors and epithelial cells in preventing self-determined cell death, the effect of changes in the matrix seen in vivo will be a summation of the different signals received.
In summary, therefore, we have defined a key role for matrix in the protection of airway epithelial cells from apoptosis. Airway epithelial cells are able to synthesize their own matrix that contributes to this survival signal. Changes in the matrix factors present within the airway wall during inflammation may alter survival signaling in both airway epithelial cells and other important structural cells in the airway. Indeed, this may represent a generic protective mechanism designed to maintain the integrity of the airway wall during chronic inflammatory insults.
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ACKNOWLEDGMENTS |
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GRANTS
This work was funded by a Biotechnology and Biological Sciences Research Council Cooperative Awards in Science and Engineering studentship in association with Aventis Pharmaceuticals.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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