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Growth factors but not gap junctions play a role in injury-induced Ca²⁺ waves in epithelial cells

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

View larger version (33K): [in a new window]   Fig. 1. Injury induces an intercellular Ca2+ wave. Cells were loaded with 4 µM fluo-3 AM for 30 minutes and washed two times. An initial baseline image is shown in (A). In response to a circular wound 250 µm in diameter made at 76 seconds (asterisk), cells immediately adjacent to the injury site displayed an increase in intracellular Ca2+ (B). The elevation in Ca2+ propagated as a radial pattern to neighboring cells (C-F). The response diminished over time and the intracellular Ca2+ levels of most cells decreased to original background levels within minutes (G). Intensity scale is shown in A, with red indicating highest Ca2+ levels and blue indicating lowest Ca2+ levels. The horizontal white bar in A represents 50 µm. Images are representative of 30 independent experiments. This series of images is taken from Movie 1 (http://jcs.biologists.org/supplemental).

View larger version (17K): [in a new window]   Fig. 2. Representative plot of the change in fluorescence that occurs after injury. The average fluorescence of a 512 µm x 512 µm field of cells was recorded every 789 milliseconds. Background fluorescence, F0, was subtracted and the percentage change in fluorescence with respect to F0 was calculated for each time point and plotted as described in Materials and Methods. The increase in fluorescence is immediate and peaks within 15 seconds of injury and then diminishes more slowly over time to original background levels within 100 seconds. The graph is labeled with letters that refer back to the images shown in Fig. 1.

View larger version (33K): [in a new window]   Fig. 3. EGF initiates a delayed Ca2+ response in non-wounded cultures. Cells were loaded with fluo-3 AM, washed and background images collected. 25 ng/ml of EGF was added, and the response was monitored for one minute. A response was not detected until 30 seconds after EGF addition (arrows). The horizontal white bar in the top figure represents 50 µm. The intensity scale is also shown, with, red indicating the highest Ca2+ levels, and blue indicating the lowest Ca2+ levels. Images are representative of 10 independent experiments.

View larger version (24K): [in a new window]   Fig. 4. EGF elicits a specific Ca2+ response. (A) A series of concentrations of EGF were evaluated, with images acquired every 789 milliseconds and data plotted as a percentage change in average fluorescence. The darker lines represent the response to EGF. The higher the concentration of EGF, the higher the number of cells recruited and the faster the response time. Cells incubated in 1 µM tyrphostin AG1478 for two hours before EGF addition did not respond. HEPES-buffered saline, lacking any growth factor (negative control) or containing 25 ng/ml PDGF or FGF-2, did not induce a detectable response. Images are representative of three independent experiments. (B) HCE-Ts migrated to EGF, with a maximal response seen at a concentration of 10 ng/ml EGF. In contrast, the same cells did not migrate to PDGF within the concentration range of 0.1 to 100 ng/ml PDGF. One representative experiment is shown in which the number of cells migrating per 10x field (average of 10 fields) was calculated (+/–, s.d.). Cells did not migrate in the absence of growth factor (negative control, (–)), but migrated extensively in the presence of 10% FBS (positive control, (+)).

View larger version (20K): [in a new window]   Fig. 5. EGF augments the injury-induced Ca2+ wave. Percentage change in average fluorescence is plotted for cells pre-treated in the presence or absence of 50 ng/ml EGF or tyrphostin AG1478. (A) Cells pretreated with EGF 90 seconds before injury produce a wave that is enhanced in duration, magnitude and peak fluorescence. (B) Cells pretreated with 1 µM tyrphostin AG1478 did not respond to EGF but did display an injury-induced wave. The graph is representative of three individual experiments.

View larger version (51K): [in a new window]   Fig. 6. Characterization of the source of Ca2+ during injury. (A) A schematic diagram of the wound model, illustrating that the cells located along the immediate wound edge respond first (left image), followed seconds later by propagation of a Ca2+ wave to neighboring cells (right image). For B and C, the average fluorescence of individual cells at various distances from the injury site (asterisk) was recorded and plotted as a percentage change in average fluorescence. (B) Absence of extracellular Ca2+ does not inhibit propagation of the wave but prevents an injury response, depicted as an increase in intracellular Ca2+, for cells immediately adjacent to the injury site. (C) Cells treated with 1 µM thapsigargin do not exhibit propagation of a Ca2+ wave upon injury (asterisk). Only cells immediately adjacent to the injury site show release of Ca2+. The images in B and C are taken from Movie 2 and Movie 3, respectively (http://jcs.biologists.org/supplemental). The horizontal white bar represents 50 µm. The intensity scale is shown, with red indicating the highest Ca2+ levels and blue indicating the lowest Ca2+ levels. Images are representative of five individual experiments.

View larger version (39K): [in a new window]   Fig. 7. Identification of connexins. 10 µg of protein was loaded into each lane of a 12% SDS-PAGE. Following electrophoresis, proteins were transferred to a PVDF membrane, and mouse monoclonal antibodies were used to probe for connexins 50, 43, or 32, all of which were detected. Positive lysates were run for connexins 43 and 32 to confirm identification. Known markers are indicated by their molecular mass (kDa) along the left edge. Results are representative of four independent experiments.

View larger version (27K): [in a new window]   Fig. 8. Gap junctions do not mediate propagation of the injury-induced Ca2+ wave. Cells were pretreated and loaded with fluo-3 AM, washed with HEPES-buffered saline and monitored using CLSM. (A) Neither 20 µM -GA nor 2 mM 1-heptanol prevented propagation of the Ca2+ wave. (B) Cells incubated with 5-CFDA were photobleached and followed for 30 minutes. Cortical astrocytes displayed refilling, whereas HCE-Ts did not. (C) A linear wound 50 µm wide was made within a confluent region of cells to create an acellular region (region delineated by two horizontal red lines). One hour later, background images were taken (0 s) at 10x magnification, and then a circular wound (depicted by a red oval) was made near the original linear wound (5 s). The injury-induced Ca2+ wave propagated in a normal radial pattern (10 s and 20 s), with no apparent difference in intensity or rate. The horizontal white bar represents 100 µm. Results are representative of three independent experiments.

View larger version (23K): [in a new window]   Fig. 9. Conditioned wound media induces a Ca2+ response that is inhibited with apyrase. (A) Conditioned media from injured corneal epithelial cells was added to fluo-3-AM-loaded epithelial cells and imaged using confocal microscopy. The response was inhibited when the wound media was treated with 30 U/ml apyrase for two minutes. (B) Cells respond to addition of 2.5 µM ATP or UTP with an immediate increase in [Ca2+]i. Percentage change in average fluorescence was calculated and plotted. (C) No elevation in intracellular Ca2+ was detected when ATP was added to non-wounded cells pretreated with thapsigargin. (D) Pretreatment of cells with 50 µM of the phospholipase-C inhibitor, U73122, prevented propagation of a Ca2+ wave following injury. Cells immediately adjacent to the injury site (asterisk) display an elevation in intracellular Ca2+, and the injury response is similar to that exhibited by cells pretreated with 1 µM thapsigargin (see Fig. 6C). The horizontal white bar represents 50 µm. The intensity scale is shown, with red indicating highest Ca2+ levels and blue indicating lowest Ca2+ levels. Images are representative of three individual experiments.