1 Departments of Pathology,
2 Cognetix, Inc., Ivoryton, CT 06442, USA
3 Ophthalmology and
4 Biochemistry, Boston University School of Medicine, Boston, MA 02118, USA
*Author for correspondence (e-mail: vickery{at}med-biochem.bu.edu)
Accepted August 30, 2001
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SUMMARY |
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Movies available on-line
Key words: Ca2+ signaling, Corneal epithelium, EGF, Wound repair
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INTRODUCTION |
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These therapeutic advancements stem from the observation that alterations in growth factor and growth factor receptor expression, availability and localization are normal components of the healing process. For example, our laboratory has shown that 30 minutes after injury an increase in the expression of transforming growth factor-ß1 (TGF-ß1) mRNA occurs in corneal fibroblasts along the wound edge, and this is associated with an increase in TGF-ß receptor binding. Furthermore, these elevations are enhanced by the addition of exogenous TGF-ß1 (Song et al., 2000). Matrix-associated growth factors such as TGF-ß1 and fibroblast growth factor-2 (FGF-2) have also been transiently detected along the corneal wound edge in vivo (Trinkaus-Randall and Nugent, 1998). Finally, Zieske et al., established that activation of the EGF receptor is required for epithelial cell migration and proliferation, events important for wound repair (Zieske et al., 2000).
Like many other biological processes, wound repair can not be characterized by a simple sequence of events. Evidence from numerous studies suggests that injury initiates activation and interaction of a multitude of intracellular signaling pathways, leading to induction of specific downstream events. Ca2+ is an important early messenger whose transient cytosolic oscillations can be induced by an extensive variety of external stimuli, such as binding of peptide factors to receptors in the plasma membrane or mechanical stimulation. The two main storage sites for Ca2+ are the endoplasmic/sarcoplasmic reticulum and the extracellular space, where the [Ca2+] is maintained at levels 10,000 fold higher than in the cytosol. Ca2+ concentration must normally be kept low in the cytosol because numerous Ca2+-binding proteins reside there. Ca2+ causes a change in activity of these cytosolic proteins, which include protein kinases and phosphatases that participate in regulation of the signaling pathways that determine cellular response (van Haasteren et al., 1999; Clapham, 1995; Berridge, 1997).
Several laboratories have reported that mechanical stimulation results in an immediate transient elevation in [Ca2+]i that spreads quickly as a wave to neighboring cells. Much of this work employed the type of mechanical stimulation that involves distortion of a single cell rather than gross mechanical injury resulting in rupture and/or removal of a group of cells. Some of the cell types studied include articular chondrocytes (Grandolfo et al., 1998), glial cells (Charles et al., 1991), pancreatic islet cells (Cao et al., 1997; Bertuzzi et al., 1999), osteoblastic cells (Jorgensen et al., 1997), and a variety of epithelial cells, including those originating from lens (Churchill et al., 1996), airway (Hansen et al., 1993), liver (Frame and de Feijter, 1997) and mammary (Enomoto et al., 1994) tissue. Investigators have focused on the underlying mechanisms for the multicellular Ca2+ wave, and some have suggested that it propagates via gap junctions, which are aggregates of intercellular channels comprised of connexins (Goodenough et al., 1996). Unlike the growth factor and growth-factor receptor changes mentioned previously, this Ca2+ response is immediate and initiated within milliseconds of injury. The study of this early and rapid event was facilitated by the use of confocal microscopy or rapid digital imaging microscopy in combination with special dyes that fluoresce upon Ca2+ binding (Grynkiewicz et al., 1985). Investigation of these early events may help decipher activation and regulation of the later events and final outcome of wound repair.
This paper characterizes the cellular Ca2+ response to injury in corneal epithelial cells. The cornea is a protective structure and offers an ideal system for the study of wound repair, because it is a simplified avascular and transparent tissue. It is divided into three regions, which are, from anterior to posterior, a squamous columnar epithelium that is multilayered, a sparsely populated stroma with a highly organized matrix and an endothelial monolayer (Trinkaus-Randall, 2000). Injury-induced Ca2+ waves have not been studied in the corneal epithelium and, unlike most previous studies, we chose to injure confluent monolayers of cells in a gross manner to more closely approximate an actual wound. We imaged cells at a rapid rate (one image every 789 milliseconds) to capture the responses of this immediate change. Although growth factors are important in wound repair, the effect of growth-factor addition on the injury-induced Ca2+ wave has not been evaluated to date.
This study describes the cellular Ca2+ response of epithelial cells to mechanical injury, with or without the addition of exogenous growth factors, identifies the source of Ca2+ for the response and proposes a mechanism by which the signal may spread to neighboring cells. Addition of EGF, but not PDGF-BB (at the same concentration), resulted in increased [Ca2+]i, and EGF specifically enhanced the amplitude and duration of the injury-induced Ca2+ wave. We found that propagation of the wave was dependent on intracellular Ca2+ stores and was not mediated via gap junctions. We show evidence that propagation of the injury-induced wave occurs via diffusion of an extracellular mediator, such as the nucleotide ATP or UTP, that is released upon cell damage.
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MATERIALS AND METHODS |
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Antibodies and reagents
All mouse monoclonal and rabbit polyclonal antibodies against connexin types 32, 43 and 50 were purchased from Zymed Laboratories, Inc. (South San Francisco, CA). The fluorescent Ca2+ indicator fluo-3 AM and pluronic acid were from Molecular Probes, Inc. (Eugene, OR), as was the 5-carboxyfluorescein diacetate (5-CFDA) AM used in the photobleaching experiments. Thapsigargin, BAPTA/AM and tyrphostin AG1478 were obtained from Calbiochem (La Jolla, CA). The gap-junction inhibitors 1-heptanol and 18-glycyrrhetinic acid (18
-GA) were bought from Sigma Chemical Company (St. Louis, MO), as were the nucleotides adenosine triphosphate (ATP) and uridine triphosphate (UTP), CdCl2 and the phospholipase C inhibitor, U73122. EGF was purchased from Gibco BRL/Life Technologies, whereas the other growth factors (PDGF-BB and FGF-2) were from R & D Systems (Minneapolis, MN).
Confocal microscopy
Epithelial cells grown to confluency on 25 mm round glass (No. 1) coverslips were quiesced for 18 to 24 hours before experimentation in Keratinocyte-SFM lacking BPE and EGF. For all experiments, cells were incubated in an HEPES-buffered saline solution containing 137 mM NaCl, 5 mM KCl, 4 mM MgCl2, 3 mM CaCl22H2O, 25 mM glucose and 10 mM HEPES (Cornell-Bell et al., 1990b). Cells were incubated in 4 µM fluo-3 AM, a dye that fluoresces upon binding Ca2+, supplemented with 0.02% pluronic acid in DMSO for 30 minutes at 37°C. After rinsing two times in HEPES-buffered saline, the live cells were placed in an open chamber (Molecular Probes, Inc., Eugene, OR) with 500 µl of HEPES solution and positioned on the stage of a Zeiss LSM 510 Axiovert confocal laser scanning microscope equipped with an Argon laser. For each experiment, cells were scanned for at least five to 10 seconds before the addition of growth factor and/or injury to establish a base line fluorescence reading. All perturbations were made while continuously scanning the cells every 789 milliseconds. A circular wound 200 to 400 µm in diameter was made and the response was recorded for a maximum of 200 seconds. To prevent the influx of extracellular Ca2+, cells were incubated in either a Ca2+-free HEPES-buffered saline solution (137 mM NaCl, 5 mM KCL, 4 mM MgCl2, 1 mM EGTA, 25 mM glucose and 10 mM HEPES) for 30 minutes prior to use or were incubated in 10 µM CdCl2 for 10 minutes prior to wounding. To prevent the utilization of intracellular Ca2+ stores, cells were incubated with 1 µM thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-ATPase, for 30 minutes prior to imaging. To determine whether gap junctions were functioning, cells were incubated in either 2 mM 1-heptanol or 20 µM 18-GA for 30 minutes before injury. In addition, because photobleaching has been demonstrated to be one of the most reliable methods for determining the presence of functioning gap junctions (Lee et al., 1994), 5-CFDA AM-loaded cells (5 µM) were bleached using the Argon laser and then followed for refilling over 30 minutes.
Image analysis
To evaluate Ca2+ dynamics over time, changes in the average fluorescence of the entire field of cells being imaged was plotted over time. The LSM 510 Imaging Software was used to determine average fluorescence of a 512 µm x 512 µm field for each 789 millisecond time point. The data were transferred to Microsoft Excel to perform calculations and plot the graph. To calculate the percentage change in average fluorescence with respect to the first time point (F0) reading, the following equation was applied to each 789 millisecond time point (F):
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The results were then plotted. For experiments in which cells were wounded, the cleared region was not included when calculating average fluorescence of the field. When Ca2+ oscillations of individual cells were plotted over time, the specific region of interest, that is, the cell, was outlined and the equation shown above used for calculating percentage change in the average fluorescence of that cell.
Migration
Epithelial cells grown to confluency on 100 mm tissue culture dishes were quiesced for 18 hours before use. Migrations were performed using Costar Transwell inserts (6.5 mm diameter) containing tissue-culture-treated polycarbonate membranes (8 µm pore size). Binding buffer was used for both diluting growth factors and resuspending the cells and was prepared as follows: 0.05% gelatin and 25 mM HEPES in KSFM. 600 µl of binding buffer, with or without growth factor, was added to each of the bottom chambers. Pure binding buffer was used as a negative control for migration, whereas 10% FCS in binding buffer was used as a positive control. Trypsinized cells were pelleted and resuspended in a volume of binding buffer that resulted in a final concentration of 125,000 cells/100 µl, and 100 µl of the cell suspension was added to each of the top chambers. The migration was carried out at 37°C and, after eight hours, migrated cells were fixed with methanol for 10 minutes at room temperature. Nonmigrated cells were swabbed from the tops of the membranes, and after permeabilizing the migrated cells with 0.1% Triton X-100 in PBS for one minute they were stained with 5 µg/ml propidium iodide (Molecular Probes, Eugene, OR) for 10 minutes at room temperature. The polycarbonate membranes were removed and mounted onto glass slides with a drop of SlowFade Antifade (Molecular Probes, Eugene, OR). For each membrane, the total number of cells was counted in each of 10 random fields (one field covering an area of 0.37 mm2) and an average and standard deviation calculated.
Western blot analysis
To determine whether connexins were present, cells were cultured to confluency in 100 mm tissue culture dishes and washed once with PBS. Lysis buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris, 1 mM EGTA, 0.5% N P-40, 2 mM PMSF, 2 mM Na3VO4) was added and the cells were removed using a cell scraper. The cell lysate was sheared using a syringe fitted with a 20 gauge needle and then centrifuged for five minutes. The supernatant was collected and protein content was determined using the BCA protein assay (Pierce, Rockford, IL) following the manufacturers instructions. Electrophoresis was performed using 12% SDS-polyacrylamide mini-gels (1.5 mm thickness). 10 µg of protein was loaded per lane and compared to positive control cell lysates (for connexins 32 and 43) (provided by Zymed Laboratories, Inc.). Proteins were transferred to a PolyScreen PVDF membrane (NEN Life Science Products, Inc., Boston, MA) and nonspecific binding was blocked by incubating the membranes in 5% BSA in TBST overnight at 4°C. Membranes were probed with a primary antibody of the appropriate concentration in TBST containing 1% BSA for one hour at room temperature with gentle agitation. After washing the membranes three times (10 minutes each) in 1% BSA in TBST, they were incubated with horseradish-peroxidase-conjugated secondary antibody in TBST containing 1% BSA (Amersham Pharmacia Biotech, Piscataway, NJ). After washing the membrane with TBST, the chemiluminescence enzymatic reaction was carried out according to the manufacturers instructions (NEN Life Science Products, Inc., Boston, MA): the blots were exposed to film and the film was developed.
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RESULTS |
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Evidence for release of an extracellular soluble mediator upon mechanical injury
As gap junctions did not participate in the injury-induced Ca2+ wave, we tested the hypothesis that an extracellular soluble mediator was released upon injury and diffused away from the injury site. In one set of experiments, conditioned media was collected from a group of HCE-Ts within seconds of the injury. When this media was added to a separate group of fluo-3-AM-loaded HCE-Ts, a significant increase in fluorescence (35%) was observed for all of the cells in the field (Fig. 9A). Because nucleotides such as ATP function as extracellular messengers, a parallel experiment was conducted where the conditioned wound media was incubated in the presence of 30 U/ml apyrase before addition to the cells. The addition of apyrase to the media inhibited any detectable elevations in intracellular Ca2+ (Fig. 9A). In addition, media collected immediately from injured cells possessed a 3.5 fold increase in the amount of extracellular ATP over control uninjured cells as determined using a luciferase assay.
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DISCUSSION |
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Our goal was to evaluate Ca2+ signaling resulting from mechanical injury to determine how Ca2+ wave propagation is mediated. Creating a circular wound 200 to 400 µm in diameter resulted in an immediate transient intercellular Ca2+ wave that traveled away from the injury site. The source of Ca2+ was determined to be both extracellular and intracellular (ER) stores. Specifically, when cells were incubated in Ca2+-free HEPES-buffered saline the increase in [Ca2+]i at the wound edge depended on extracellular Ca2+ potentially entering through leaky membranes created by physical injury or by the opening of stretch-activated Ca2+ channels in the plasma membrane. However, the presence of the Ca2+ wave under these extracellular Ca2+-free conditions suggests that wave propagation relies on intracellular Ca2+ stores and that the propagation is not dependent on [Ca2+]i changes that occur in cells at the immediate wound edge. The fact that the mechanically stimulated cells obtain extracellular Ca2+ whereas the wave relies on intracellular Ca2+ is consistent with theories that the injury response is a two-phase process (Boitano et al., 1994; Sanderson et al., 1994; Hansen et al., 1995; Frame and de Feijter, 1997; Venance et al., 1997).
One potential mechanism for wave propagation is via diffusion of an intracellular mediator through gap junction complexes. This mechanism occurs in a variety of cell types, including articular chondrocytes (DAndrea and Vittur, 1996), airway epithelial cells (Hansen et al., 1993; Boitano et al., 1994), glial cells (Charles et al., 1992; Venance et al., 1997), smooth muscle cells (Christ et al., 1992; Young et al., 1996) and endothelial cells (Drumheller and Hubbell, 1991). There is some controversy as to whether Ca2+ or another messenger, such as IP3, passes through gap junctions to create the wave (Boitano et al., 1992; Sanderson et al., 1994; DAndrea and Vittur, 1997; Jorgensen et al., 1997; Grandolfo et al., 1998). However, evidence for a second mechanism emerged when it was demonstrated that waves could cross acellular regions and that fluid flow could affect propagation of the wave (Enomoto et al., 1992; Hassinger et al., 1996; Sammak et al., 1997; Guthrie et al., 1999). Thus the injury could mediate a signal that induced diffusion of an extracellular messenger released from the damaged cells. Interestingly, propagation via this mechanism was discovered in some cells that lacked gap junctions (Frame and de Feijter, 1997). Furthermore, there is evidence in some culture systems that both mechanisms co-exist, simultaneously and independently (Enomoto et al., 1994; Frame and de Feijter, 1997; Sammak et al., 1997; Braet et al., 2001).
Our results suggest that the Ca2+ wave in HCE-Ts or primary epithelial cells does not require gap junctions but depends on a diffusable extracellular factor. This conclusion is based on experiments we performed using gap junction inhibitors, FRAP analysis and wave propagation across an acellular region (Fig. 8). Even incubation in EGTA for 30 minutes, which resulted in rounding-up of the cells and loss of cell-cell contact, did not inhibit wave propagation. Although functioning gap junctions are known to be present in the corneal epithelium in vivo (Dong et al., 1994; Williams and Watsky, 1997), several endogenous and exogenous factors, such as low or high [Ca2+]i, may modulate intercellular communication through gap junctions in cultured cells (Goodenough et al., 1996; Jansen et al., 1996; Frame and de Feijter, 1997).
The identification of the extracellular messenger is currently being investigated. A number of soluble factors produced by cells mobilize Ca2+, among them nucleotides, glutamate, acetylcholine and growth factors. ATP causes Ca2+ waves in glial cells (Centemeri et al., 1997; Guthrie et al., 1999), mast cells (Osipchuk and Cahalan, 1992), osteoblasts (Jorgensen et al., 1997), chondrocytes (DAndrea and Vittur, 1996), airway epithelial cells (Hansen et al., 1993) and mammary epithelial cells (Enomoto et al., 1994). Corneal epithelial cells used in this study responded to both ATP and UTP, and our experiments and those of others using apyrase suggested ATP as a potential candidate (Guthrie et al., 1999). ATP can function as an extracellular messenger for a variety of cellular processes in both excitable and nonexcitable cells (Burnstock, 1997; Neary et al., 1999). By binding the P2Y purinergic receptor, ATP initiates an intracellular signaling cascade that begins with the synthesis of IP3 by the action of phospholipase C and then release of Ca2+ from intracellular storage sites (Harden et al., 1995; North and Barnard, 1997). This IP3-mediated mechanism is consistent with our results showing that thapsigargin inhibits propagation of the Ca2+ wave. In addition, our results suggest that an extracellular messenger is released upon cell damage. But in cases where cell damage does not occur, active secretion of the messenger must occur. This has important implications for understanding whether the wave is generated by release of the messenger from a single point source or by a regenerative mechanism in which there is sequential secretion of the messenger from cells along the path of the Ca2+ wave (Osipchuk and Cahalan, 1992; Sneyd et al., 1994; Frame and de Feijter, 1997; Guthrie et al., 1999).
It will also be important to coordinate the knowledge obtained from these studies with the information that growth factors such as EGF play a significant role in epithelial wound repair (Leibowitz et al., 1990; Moulin, 1995; Steed, 1998). Addition of EGF to control HCE-Ts results in elevation of [Ca2+]i as the EGF diffuses across the field, but this response is not immediate (Fig. 3) and may take 20 to 40 seconds to detect. This delay may be due to the fact that receptor dimerization is required for EGF-receptor activation, resulting in the release of intracellular Ca2+ stores through a phospholipase C/IP3-mediated mechanism. In contrast, we demonstrated that rapid propagation of the injury-induced Ca2+ wave is independent of the activation of EGF receptors by wounding cells after they were incubated in tyrphostin AG1478 (Fig. 5B).
Although activation of the EGF receptor is not required for wave propagation, there is a synergistic relationship between the addition of EGF and the amplitude of the Ca2+ wave when the wound is made during EGF-induced [Ca2+]i elevation. The fact that the Ca2+ response to EGF cannot be mimicked by other growth factors that bind tyrosine kinase receptors, such as PDGF-BB, suggests some specificity. Furthermore, lacritin, a novel secretion enhancing factor isolated from the lacrimal gland, also induces a distinct wave form in corneal epithelial cells that is not a typical target wave (Sanghi et al., 2001). This is important when one considers that the corneal epithelium is bathed in tear fluid, rich in a variety of soluble factors, many of which become elevated following corneal injury (Sheardown and Cheng, 1996; Vesaluoma et al., 1997a; Vesaluoma et al., 1997b). Other studies in our laboratory have concluded that addition of exogenous EGF results in faster wound closure as well as in an increase in integrin ß4 receptor in cultured corneal epithelial cells (Song et al., 2001). We believe that this result provides evidence that enhancement of the injury-induced early Ca2+ response by the presence of EGF may have important longer term effects on wound repair. This may be especially important for certain pathologies, such as diabetes, in which cells are shown to be less responsive to growth factors (Embil et al., 2000; Ladin, 2000).
Finally, it is important to consider the biological significance of an elevation in [Ca2+]i that travels as a wave. There are numerous cytosolic proteins that alter their activity after binding to Ca2+. In addition to cell type and surrounding environment, the timing, duration, frequency and amplitude of Ca2+ oscillations most probably play a role in determining which specific signaling pathways are activated following injury. Signaling pathways that lead to changes in expression, localization or activity of proteins involved in cellular adhesion, migratory ability and proliferation are candidates to be investigated. Studies have shown that Ca2+ may regulate motility (Brundage et al., 1991; Gilbert et al., 1994), proliferation (Byron and Villereal, 1989; Wahl and Gruenstein, 1993; Means, 1994), differentiation, and secretion (Marks and Maxfield, 1990). We have also shown that initial contact of corneal epithelial cells with a substrate is accompanied by Ca2+ oscillations (Trinkaus-Randall et al., 2000), and these results are supported by the more recent adhesion studies of Juliano (Short et al., 2000). The data presented and discussed here suggest a potentially significant role for the Ca2+ wave in coordinating the cellular processes important for wound repair.
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ACKNOWLEDGMENTS |
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