1 Department of Anatomy and Cell Biology, Wayne State University School of Medicine, Detroit, MI, USA
2 Department of Ophthalmology, Kresge Eye Institute, Wayne State University School of Medicine, Detroit, MI, USA
*Author for correspondence (e-mail: mkurpaku{at}med.wayne.edu)
Accepted August 7, 2001
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SUMMARY |
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Key words: Apoptosis, Corneal epithelium, Hypoxia, Laminin 5
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INTRODUCTION |
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In epithelial cells a diversity of signals and events can induce the apoptotic form of cell death, including disruptions in normal cell-extracellular matrix (ECM) interactions. Apoptosis specifically induced by a breach in cell-matrix adhesion has been termed anoikis (Frisch and Francis, 1994). In stratified epithelia such as skin, tongue and cornea, an adhesion complex mediates cell-extracellular matrix attachment. The structure consists of a cytoplasmic hemidesmosome, extracellular anchoring filaments (composed of laminin 5) and anchoring fibrils (composed of collagen type VII), and the basement membrane (Borradori and Sonnenberg, 1999). 6ß4 integrin is physically associated with the hemidesmosome and is unique in that its cytoplasmic domain interacts with intermediate filaments as opposed to actin (Stepp et al., 1990). Laminin 5 (
3ß3
2) is a major adhesive ligand resident in the basement membrane and can function as an extracellular attachment protein for epithelium. It binds with high affinity to
6ß4 integrin (Champliaud et al., 1996).
Laminin 5/6ß4 interactions are crucial for the assembly and maintenance of adhesion complex components because the protein pair forms the core of the hemidesmosome (Green and Jones, 1996). The loss of hemidesmosomes in basal epidermal cells of transgenic mice lacking functional ß4 integrin correlated to weak adhesion and cell degeneration attributed to apoptosis (Dowling et al., 1996). The targeted disruption of the Lama3 gene, which encodes the
3 subunit of laminin 5, resulted in survival defects in homozygous null animals (Ryan et al., 1999). These authors (Dowling et al., 1996; Ryan et al., 1999) have suggested that laminin 5 provides a survival advantage for keratinocytes and that
6ß4 integrin interacts with laminin 5 to mediate an unidentified signal essential for cell survival.
Hypoxia-mediated cell death had been presumed to occur by necrosis (Jozsa et al., 1981). Recent studies however have suggested that hypoxia can also induce apoptosis in epithelial cells (Bossenmeyer-Pourie and Daval, 1997; Volm et al., 1999). Upregulation of collagen type IV and fibronectin observed as a consequence of chronic hypoxia (Vyas-Somani et al., 1996; Kim et al., 1996; Berg et al., 1998) implies that cell-matrix adhesion may not be affected by available oxygen. By contrast, acute hypoxia resulted in a significant downregulation of cell surface integrins, CD44 and NCAM, and an associated decrease in cell adhesion in two human melanoma cell lines and a human adenocarcinoma line (Hasan et al., 1998). In human keratinocytes subjected to acute hypoxia the number of cell surface integrin receptors did not decrease, but laminin 5 secretion was significantly inhibited (OToole et al., 1997).
We hypothesize that a disruption in functional laminin 5 protein in the extracellular matrix of hypoxic corneal epithelial cells promotes apoptosis. To test this hypothesis, we analyzed the effect of hypoxia on corneal epithelial cell proliferation, programmed cell death and laminin 5 production. Our work demonstrates that human corneal epithelial cells subjected to chronic hypoxia undergo apoptosis and deposit less functional laminin 5 protein into the extracellular matrix. We further examined the role of cell-matrix integrity in hypoxia-induced apoptosis by disturbing adhesion with function blocking antibodies to laminin 5. Our observations suggest that compromised cell-matrix communication, via altered homeostatis of laminin 5 function, is one mechanism of hypoxia-induced apoptosis.
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MATERIALS AND METHODS |
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Culture of human corneal epithelial cells
The transfected human corneal epithelial cell line 10.014 pRSV-T, referred to as HCE-T (Kahn et al., 1993) was a generous gift from S. Ward (The Gillette Company, Gillette Medical Evaluation Laboratories, Gaithersburg, MD). HCE-T were maintained in serum-free keratinocyte growth medium containing 0.1% bovine insulin, 0.1% human epidermal growth factor, 0.4% bovine pituitary extract and 0.1% hydrocortisone (KGM; Clonetics , San Diego, CA). HCE-T are viable until passage 20, in this study the cells were used between passage 16 and 17. Human corneal epithelial cells (HCEC) were obtained as cryopreserved tertiary cultures from young donors (Cascade Biologics, Portland, OR). HCEC were maintained in serum-free EpiLife medium containing human corneal growth supplement as supplied by Cascade. These cells were not passaged further, but were used as tertiary cultures for all studies.
For studies analyzing the effect of oxygen on cell behavior, one set of cells was maintained at 37°C and 5% CO2 in a conventional humidified tissue culture incubator. The oxygen level under these conditions (20%) was defined as normoxic. A second set of cells was maintained at 37°C and 5% CO2 in a humidified environmental chamber (Coy Laboratory Products, Ann Arbor, MI). The oxygen level under these conditions (2%) was defined as hypoxic. An oxygen analyzer was used to maintain the oxygen level at 2% by regulating the flow of a calibrated mixture of 95% nitrogen and 5% CO2 into the chamber. Cells were maintained for up to 14 days depending on the experiment, with medium changes every other day.
Cell proliferation assay
HCE-T were seeded in 96-well uncoated tissue culture plates at an initial plating density of 2000 cells/well and incubated in either 2% or 20% oxygen for a total of 1, 3, 7, 10, 12 or 14 days. In a second experiment cells were incubated in 20% oxygen in the presence of 10 µg/ml mAb P3H9-2 to inhibit laminin 5 function. Cells were cultured for a total of 3 days, including treatment with the antibody for 2 days. As a control, cells were treated with 10 µg/ml IgG. Four hours before analysis on each of the indicated days 5-bromo-2'-deoxyuridine (BrdU) was added to each of the wells to a final concentration of 10 µM. Cells were fixed and DNA was denatured using reagents supplied with a cell proliferation ELISA kit (Roche Diagnostics, Indianapolis, IN). Cells were labeled with anti-BrdU peroxidase conjugate, washed, and incubated with color development substrate containing tetramethylbenzidine. The absorbance of the samples was measured at 450 nm wavelength (A450). The result of the ELISA is represented as a graph of the mean absorbance (A450)±s.e.m. (in arbitrary units) as a function of time in culture and oxygen level. Thirty replicate wells of each time point and oxygen level were assayed for the ELISA (n=30). Statistical analysis using Students two-tailed t-test with significance level of P<0.05 was used to compare proliferation as a function of time in culture and oxygen level.
Detection of apoptosis in cultured human corneal epithelial cells
Apoptosis in HCEC adherent to glass coverslips was assayed by determining mitochondrial integrity using the MitoLightTM Apoptosis Detection Kit (Chemicon). Cells were cultured in 2% or 20% oxygen for 3, 5 or 7 days. In a second experiment, cells were cultured in 20% oxygen in the presence of 10 µg/ml mAb P3H9-2 to block laminin 5 function. Cells were cultured for a total of 3 days including treatment with the antibody for 2 days. As controls, cells were cultured in the presence of 10 µg/ml control IgG. At each culture time point unfixed cells were incubated with the MitoLightTM reagent for 30 minutes at 37°C, as suggested by the kit protocol. The cells were placed on a microscope slide and observed immediately using a Zeiss Axiophot fluorescence microscope.
In healthy cells, the lipophilic cationic dye employed in the assay partitions to the cytoplasm and also accumulates in mitochondria, owing to its uptake by biochemically intact organelles. In apoptotic cells with altered mitochondrial membrane potential the dye is evenly distributed throughout the cytoplasm. Using filters to detect fluorescein and rhodamine, healthy cells are identified as containing red mitochondria against a green background of cytoplasmic dye. We scored any cell with at least one labeled mitochondria as viable. By contrast, apoptotic cells are uniformly green with no detectable red-labeled mitochondria. Identical fluorescein and rhodamine fields at 20x magnification were digitized (SPOT Diagnostics, Sterling Heights, MI) and the digital images were overlaid using MetaMorph software (Universal Imaging, West Chester, PA). Five images were captured per coverslip. The total number of cells per field and the total number of cells lacking any red-labeled mitochondria (i.e. fluorescent green only) were counted to calculate the percentage of apoptotic cells as a function of culture conditions. Statistical analysis was performed using Students two-tailed t-test and significance level P<0.05.
To confirm apoptosis, human corneal epithelial cells cultured for 7 days in 20% or 2% oxygen were lysed in gel sample buffer containing 60 mM Tris-HCl pH 6.8, 8 M urea, 1% SDS, 1% glycerol and 0.5% ß-mercaptoethanol (Klatte et al., 1989). Protein concentrations were determined by the method of Henkel and Beiger (Henkel and Beiger, 1994) and 15 µg total cell protein were resolved using 7.5% SDS-PAGE. The separated proteins were electrophoretically transferred to nitrocellulose membrane and processed for western blot analysis using mAb to PARP at 1:1000 dilution. Immunoreactive proteins were detected and analyzed as described in the following section.
Western blot analysis of intracellular and extracellular laminin 5 content in cultured human corneal epithelial cells
HCE-T were cultured for 3 or 7 days in 2% oxygen or 20% oxygen. Cells and their deposited extracellular matrix were lysed in the urea/SDS gel sample buffer. 15 µg total cell protein was resolved using 7.5% SDS-PAGE. The separated proteins were electrophoretically transferred to nitrocellulose membrane and processed for Western blot analysis using mAb D4B5 to laminin 2 chain (1:1000 dilution).
Immunoreactive proteins were detected using alkaline phosphatase-conjugated goat-anti mouse IgG (1:3000) as the secondary antibody coupled with the Immun-Star chemiluminescent protein detection system (BioRad Laboratories, Hercules, CA). Molecular weight markers obtained from BioRad were run with each gel and used to approximate the molecular weights of the immunoreactive proteins. The intensity of individual immunoreactive protein bands was determined by scanning the developed X-ray films and measuring the optical density and area of the bands using ImageQuant software (Molecular Dynamics, Sunnyvale, CA). Results of the densitometric analyses are represented in arbitrary units of the relative density of immunoreactive protein bands per µg total protein loaded on the gel. Western blot analysis was repeated five times with similar results.
Western blot analysis of ECM deposited by human corneal epithelial cells
HCE-T, in wells of a 24-well tissue culture plate, were cultured at an initial plating density of 25,000 cells/well in 2% or 20% oxygen for 7 days. Cells were exposed to the different oxygen levels immediately following trypsinization and replating. The confluent epithelial cell layer was lysed in 0.1 N ammonium hydroxide (Langhofer et al., 1993). The ECM remaining on the culture substrate was washed extensively with sterile phosphate-buffered saline to remove all cell debris. 40 µl of gel sample buffer was used to solubilize the ECM in one well and the samples were loaded on to three different lanes of a 7.5% acrylamide gel. ECM from hypoxic and normoxic cell culture was loaded side-by-side to allow for a direct comparison of matrix composition. Western blot analysis using mAb 10B5 (1:3), Clone 17 (1:1000) or D4B5 (1:500), and densitometric analysis was completed as described. This western blot analysis was repeated five times.
Immunofluorescence microscopy
To prepare HCE-T for immunofluorescence microscopic analysis, cells cultured on glass coverslips were fixed for 5 minutes in acetone at 20°C then washed in PBS for an additional 5 minutes. The fixed cells were probed with mAb GB3 following standard single-label direct immunofluorescence techniques (Klatte et al., 1989). The cells were examined using a Zeiss Axiophot fluorescence microscope. Fluorescein images at 20x magnification were captured and digitized
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RESULTS |
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After 5 days in culture, normoxic cells still contained numerous functional mitochondria (Fig. 2C). However, apoptotic cells were now evident in the hypoxic cultures (Fig. 2D). Quantitative analysis of HCEC demonstrated a statistically significant difference in the number of apoptotic cells in normoxic versus hypoxic cultures after 5 days. In 20% oxygen 18% of HCEC were apoptotic. By contrast in 2% oxygen, 30% of the cells were apoptotic (average of five fields counted, P<0.01 compared with normoxic control).
After 7 days cells cultured under normoxic conditions still contained labeled mitochondria, with little evidence of extensive apoptosis (Fig. 2E). Indeed, the average percentage of apoptotic HCEC remained at 18%. However, extensive apoptosis was characteristic of hypoxic cells maintained in culture for 7 days (Fig. 2F). 70% of hypoxic HCEC were determined to be apoptotic at this time point in culture (average of five fields counted, P<0.01 compared with normoxic control).
To provide a quantitative analysis of apoptosis the degradation of PARP from the full-length 115 kDa to the 89 kDa fragment was assessed using western blot analysis. Although it was possible to visualize increased apoptosis using the mitochondrial assay in cultured corneal epithelial cells after 5 days, by densitometry increased PARP cleavage in hypoxic cells was not demonstrated until 7 days in culture (Fig. 3). At 7 days in culture, the 89 kDa degradation product accounted for 17% of the total PARP in normoxic cells, in contrast to 37% total PARP in hypoxic cells. An increase in the relative amount of the degraded PARP species in the hypoxic cell lysate suggests that a greater proportion of cells were apoptotic compared with normoxic controls.
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The 2 chain of laminin 5 is extracellularly processed from 155 kDa to 105 kDa (Matsui et al., 1995; Goldfinger et al., 1998). Giannelli et al. (Giannelli et al., 1999) have also suggested that further processing to 80 kDa may occur. In HCE-T lysates containing both cellular and extracellular laminin 5 protein, all three molecular weight species of the
2 chain were detected by western blot. Densitometric analysis showed that the total amount of all unprocessed and processed forms of
2 chain was decreased in hypoxic HCE-T compared with normoxic HCE-T (Fig. 4). This held true for cells cultured for either 3 days (22% less total
2 chain in hypoxic cells) or 7 days (42% less total
2 chain in hypoxic cells). The ratio of unprocessed to processed forms of the chain also differed with respect to time in culture and oxygen level. After 3 days in culture, a greater proportion of
2 chain was found to be in the processed forms in the hypoxic HCE-T (75% compared with 60% for normoxic HCE-T). By contrast, by 7 days in culture less of the processed forms of the chain were detected in hypoxic HCE-T (21% compared with 36% for normoxic HCE-T).
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DISCUSSION |
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Human corneal epithelial cells appear to be resistant to deleterious effects of hypoxia for up to 5 days in culture, as we did not detect significant changes in mitochondrial function or relative percentages of apoptotic cells until day 5. PARP cleavage, characteristic of the initiation of the apoptotic pathway, was not evident until 7 days culture in the hypoxic environment. Hypoxia-mediated apoptosis occurred in human cultured trophoblasts after 24 hours in culture (Levy et al., 2000). Treatment with EGF to enhance trophoblast differentiation conferred a resistance to hypoxia-mediated apoptosis. These authors suggested that differentiated cells might be more resistant to hypoxia. However, while adult ventricular myocytes demonstrated evidence of apoptosis after 1 hour of hypoxia (de Moissac et al., 2000), neonatal ventricular myocytes became apoptotic only after 12-24 hours of hypoxia (Long et al., 1998; Tanaka et al., 1994). These results suggested that neonatal myocytes are more resistant to hypoxia. HCE-T are an SV-40 virally transformed cell line derived from adult human cornea, and HCEC are a tertiary culture derived from normal young human donors. The apoptotic response to chronic hypoxia appears to be similar in both cell types. We cannot conclude, based on the conflicting reports from other investigations, that the differentiation state of HCE-T or HCEC alone explains the ability of human corneal epithelial cells to resist the apoptotic effects of hypoxia in the short term. However, corneal epithelial cells are unique in that they typically exist and thrive in an avascular environment, deriving nutrients and oxygen from the tear film in lieu of an extensive vascular bed. Corneal cells may thus be predisposed to survival in unfavorable environments, explaining why cells in culture appear to be able to function in hypoxic conditions for relatively long periods of time compared with other cell types.
In our model, we have observed a correlation between the relative amount of processed laminin 5 2 chain and apoptosis. In HCE-T that were placed in a hypoxic environment immediately after trypsinization and replating, less
2 chain is produced after 3 days in culture. However, the relative percentage of that
2 chain that has been extracellularly processed is increased in hypoxic cells compared with normoxic cells. In an experiment not shown, HCE-T cultured for 5 days under normoxic conditions followed by 5 days under hypoxic conditions also deposited less total laminin 5
2 chain compared with cells cultured for the same amount of time in 20% oxygen only. However, a greater relative percentage of that
2 chain is in the processed form in the cells exposed to hypoxic conditions.
It has been suggested that the acquisition of a motile phenotype can alleviate anoikis (Frisch and Francis, 1994). Extracellular processing of 2 chain may be associated with the motility function of laminin 5. Taken together, between 1 and 5 days exposure to hypoxia, human corneal epithelial cells can effect an upregulation in the extracellular processing of laminin 5
2 chain in an attempt to adopt a motile phenotype and escape hypoxia-mediated apoptosis. However, the cells cannot sustain this response and at time points longer than 5 days in culture increased laminin 5
2 chain processing stops, and cells become apoptotic. Although the cells can upregulate processing of laminin 5
2 chain during short periods of hypoxic stress, the total amount of laminin 5 protein is consistently decreased. Based on our observations, we hypothesize that a decrease in total laminin 5 protein content, which in our model system occurs by 3 days in hypoxic culture, precedes the increased apoptosis (noted by mitochondrial function and PARP cleavage) observed by 7 days in culture. This suggests that the decreased laminin 5 present in the extracellular matrix of cultured human corneal epithelial cells contributes to initiation of hypoxia-mediated apoptosis.
The identity of the enzyme responsible for the extracellular processing of the laminin 5 2 chain is currently unknown. It has been suggested, though, that matrix metalloprotease 2 (MMP2) can cleave the 105 kDa
2 chain into an 80 kDa
2x species (Gianelli et al., 1999). This form of laminin 5
2 chain is correlated with a motile phenotype in breast tissue. To determine if these enzymes are involved in motile responses in corneal epithelium, we are currently determining the effect of hypoxia on MMP levels in HCE-T and HCEC.
Gonzales et al. (Gonzales et al., 1999) have shown that function-blocking antibodies to laminin 5 inhibit epithelial cell proliferation, implicating a cell signaling pathway involving laminin 5 (Gonzales et al., 1999). We show herein that hypoxia also inhibits epithelial cell proliferation. In addition, we show that hypoxia results in altered laminin 5 protein levels, and that loss of laminin 5 or function-blocking antibodies to laminin 5 upregulate apoptosis in cultured human corneal epithelial cells. The ability of cell-matrix interactions to generate cell survival signals, and the loss of those signals upon cell detachment from matrix, is the underlying mechanism of anchorage-dependent apoptosis (Meredith et al., 1993). Within corneal basement membrane, we propose that laminin 5 plays a bi-functional role in suppressing anchorage-dependent apoptosis: to ensure cell-matrix adhesion and to transmit extracellular survival signals to the nucleus. We hypothesize that one mechanism of hypoxia-mediated apoptosis in human corneal epithelium involves perturbation of the laminin 5-mediated cell signaling pathway proposed by Gonzales et al. (Gonzales et al., 1999).
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ACKNOWLEDGMENTS |
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