Migration of Human Keratinocytes in Electric Fields Requires Growth Factors and Extracellular Calcium

Currents that leak out of wounds generate electric fields lateral to the wound. These fields induce directional locomotion of human keratinocytes in vitro and may promote wound healing in vivo. We have examined the effects of growth factors and calcium, normally present in culture medium and the wound fluid, on the directional migration of human keratinocytes in culture. In electric fields of physiologic strength (100 mV per mm), keratinocytes migrated directionally towards the cathode at a rate of about 1 μm per min. This directional migration requires several growth factors. In the absence of these growth factors, the cell migration rate decreased but directionality was maintained. Epidermal growth factor alone restored cell migration rates at concentrations as low as 0.2 ng per ml. Insulin at 5–100 μg per ml or bovine pituitary extract at 0.2%–2% vol/vol also stimulated keratinocyte motility but was not sufficient to fully restore the migration rate. Keratinocyte migration in electric fields requires extracellular calcium. Changes in calcium concentrations from 3 μM to 3.3 mM did not significantly change keratinocyte migration rate nor directionality in electric fields; however, addition of the chelator ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid to migration medium reduced, and eventually abolished, keratinocyte motility. Our results show that (i) growth factors and extracellular calcium are required for electric field-induced directional migration of human keratinocytes, and (ii) keratinocytes migrate equally well in low and high calcium media. Currents that leak out of wounds generate electric fields lateral to the wound. These fields induce directional locomotion of human keratinocytes in vitro and may promote wound healing in vivo. We have examined the effects of growth factors and calcium, normally present in culture medium and the wound fluid, on the directional migration of human keratinocytes in culture. In electric fields of physiologic strength (100 mV per mm), keratinocytes migrated directionally towards the cathode at a rate of about 1 μm per min. This directional migration requires several growth factors. In the absence of these growth factors, the cell migration rate decreased but directionality was maintained. Epidermal growth factor alone restored cell migration rates at concentrations as low as 0.2 ng per ml. Insulin at 5–100 μg per ml or bovine pituitary extract at 0.2%–2% vol/vol also stimulated keratinocyte motility but was not sufficient to fully restore the migration rate. Keratinocyte migration in electric fields requires extracellular calcium. Changes in calcium concentrations from 3 μM to 3.3 mM did not significantly change keratinocyte migration rate nor directionality in electric fields; however, addition of the chelator ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid to migration medium reduced, and eventually abolished, keratinocyte motility. Our results show that (i) growth factors and extracellular calcium are required for electric field-induced directional migration of human keratinocytes, and (ii) keratinocytes migrate equally well in low and high calcium media. bovine pituitary factor human keratinocyte growth supplement keratinocyte migration medium Keratinocyte proliferation and migration into wounds are essential for cutaneous wound healing (Krawczyk, 1971Krawczyk W.S. 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In mammals, lateral fields of 10–100 mV per mm have been measured near the edge of the wounds (Illingworth and Barker, 1980Illingworth C.M. Barker A.T. Measurement of electrical currents emerging during the regeneration of amputated fingertip in children.Clin Phys Physiol Meas. 1980; 1: 87-89Crossref Scopus (109) Google Scholar;Barker et al., 1982Barker A.T. Jaffe L.F. Vanable Jr, Jw The glabrous epidermis of cavies contains a powerful battery.Am J Physiol. 1982; 242: R358-R366PubMed Google Scholar). These wound-induced electric fields and currents may promote cell migration during wound healing. We have shown previously that human keratinocytes in vitro migrate to the cathode in direct current (DC) electric fields of physiologic magnitudes, 100 mV per mm (“galvanotaxis”) (Nishimura et al., 1996Nishimura K.Y. Isseroff R.R. Nuccitelli R. Human keratinocytes migrate to the negative pole in direct current electric fields comparable to those measured in mammalian wounds.J Cell Sci. 1996; 109: 199-207PubMed Google Scholar). We have also shown that galvanotaxis of human keratinocytes in physiologic electric fields is strongest on collagen types I and IV (Sheridan et al., 1996Sheridan D.M. Isseroff R.R. Nuccitelli R. Imposition of a physiologic DC electric field alters the migratory response of human keratinocytes on extracellular matrix molecules.J Invest Dermatol. 1996; 106: 642-646Crossref PubMed Scopus (87) Google Scholar). Those galvanotaxis experiments were conducted in a commonly used serum-free keratinocyte growth medium (Boyce and Ham, 1983Boyce S.T. Ham R.G. 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Enhanced modulation of keratinocyte motility by transforming growth factor-alpha (TGF-alpha) relative to epidermal growth factor (EGF).J Invest Dermatol. 1996; 106: 590-597Crossref PubMed Scopus (134) Google Scholar). Incubation in media containing higher than 1 mM Ca++ has been shown to suppress migration (Nickoloff et al., 1988Nickoloff B.J. Mitra R.S. Riser B.L. Dixit V.M. Varani J. Modulation of keratinocyte motility.Am J Pathol. 1988; 132: 543-551PubMed Google Scholar), presumably due to production of matrix proteins and increased cell–matrix or cell–cell interactions (O’keefe et al., 1987O’keefe E.J. Briggaman R.A. Herman B. Calcium-induced assembly of adherens junctions in keratinocytes.J Cell Biol. 1987; 105: 807-817Crossref PubMed Scopus (103) Google Scholar;Kim et al., 1992Kim J.P. Zhang K. Chen J.D. Wynn K.C. Kramer R.H. Woodley D.T. 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The Keratinocyte Handbook. Cambridge University Press, Cambridge1994Google Scholar). In this study we have examined the effects of high (>1 mM) and low (<0.2 mM) extracellular Ca++ levels on keratinocyte galvanotaxis. Because we subjected cells to electric fields several hours after plating, we concentrated on the short-term direct effects of Ca++ levels on keratinocyte galvanotaxis rather than the longer-term Ca++-mediated effects, e.g., on gene expression or cell differentiation, which could also alter the migratory response. In addition, we have also examined the effects on keratinocyte galvanotaxis of growth factors normally present in the culture medium and the wound fluid. Normal human keratinocytes from neonatal foreskin epidermis were prepared and cultured following the methods described previously (Isseroff et al., 1987Isseroff R.R. Ziboh V.A. Chapkin R.S. Martinez D.T. Conversion of linoleic acid into arachidonic acid by cultured murine and human keratinocytes.J Lipid Res. 1987; 28: 1342-1349Abstract Full Text PDF PubMed Google Scholar). Aliquots of cells derived from a single donor were cryopreserved. Cells were cultured in the serum-free keratinocyte growth medium that was composed of M154 medium (Cascade Biologics, Portland, OR), 10 mM HEPES (pH 7.4), antibiotics/antimycotics (penicillin, streptomycin, and amphotericin), and human keratinocyte growth supplement (HKGS) that includes 0.18% hydrocortisone, 5 μg transferrin per ml, 0.2% vol/vol bovine pituitary extract (BPE), 0.2 ng EGF per ml, and 5 μg insulin per ml. Cultures were kept at 37°C in a humidified atmosphere of 5% CO2. Cells from two donors were used in this work, and passage 2–5 cells were used for experiments. Coverslips and cells were prepared following a procedure described previously (Sheridan et al., 1996Sheridan D.M. Isseroff R.R. Nuccitelli R. Imposition of a physiologic DC electric field alters the migratory response of human keratinocytes on extracellular matrix molecules.J Invest Dermatol. 1996; 106: 642-646Crossref PubMed Scopus (87) Google Scholar). Briefly, 12 mm glass coverslips were coated with bovine collagen I by soaking in calcium and magnesium-free phosphate-buffered saline containing 2% Vitrogen 100 (60 μg per ml) (Collagen, Palo Alto, CA) for at least 1 h at 37°C. Coverslips were rinsed three times with phosphate-buffered saline and air-dried for 5–10 min before cells were plated. Normal human keratinocytes were plated on the collagen-coated coverslips at a density of 4–6 × 104 in a 35 mm plate in keratinocyte migration medium (KMM), which is keratinocyte growth medium supplemented with 1.8 mM calcium chloride (a final Ca++ concentration is 2 mM). After 2–6 h to allow attachment, coverslips were rinsed with medium to remove unattached cells and placed in a galvanotaxis chamber. Galvanotaxis experiments were conducted for 1 h in DC electric fields of physiologic strength (100 mV per mm). If an additional factor or unique condition was being tested, coverslips were rinsed with phosphate-buffered saline twice, incubated in the medium containing the indicated factor or condition for 1 h at 37°C, then subjected to the fields in the same medium. Recombinant human EGF was obtained from Gibco Life Technologies (Grand Island, NY), human insulin from Sigma (St. Louis, MO), and bovine pituitary extract from Cascade Biologics. The free calcium concentrations in all media were later verified by using atomic absorption by Perkin-Elmer Analyst 300. The galvanotaxis chamber and apparatus for applying DC electric fields have been described previously (Erickson and Nuccitelli, 1984Erickson C.A. Nuccitelli R. Embryonic fibroblast motility and orientation can be influenced by physiological electric fields.J Cell Biol. 1984; 98: 296-307Crossref PubMed Scopus (240) Google Scholar;Nishimura et al., 1996Nishimura K.Y. Isseroff R.R. Nuccitelli R. Human keratinocytes migrate to the negative pole in direct current electric fields comparable to those measured in mammalian wounds.J Cell Sci. 1996; 109: 199-207PubMed Google Scholar;Sheridan et al., 1996Sheridan D.M. Isseroff R.R. Nuccitelli R. Imposition of a physiologic DC electric field alters the migratory response of human keratinocytes on extracellular matrix molecules.J Invest Dermatol. 1996; 106: 642-646Crossref PubMed Scopus (87) Google Scholar). KMM, or modifications as otherwise stated, was used for galvanotaxis. A physiologic electric field was applied with a constant voltage at 100 mV per mm and a current at 0.1–0.6 mA. The galvanotaxis was performed at 37 ± 2°C in room air. Cells were observed with phase contrast optics on inverted microscopes, and video images of cells were digitally captured every 10 min for 1 h to an image analysis program on a Power Macintosh 8500 using a modified version of NIH Image 1.60 and File Maker Pro 3.0. At the end of 1 h, the center of each cell was identified manually on each image. The translocation distance and directionality of migration of each imaged cell were analyzed. The translocation distance covered by each cell was measured in μm per h, and migration rate was expressed as μm per min. The directionality of the cell translocation was indicated by an average cosine, cos , of the angles that the path of each cell made with respect to the electric field direction, = ϕicos i/N (Nishimura et al., 1996Nishimura K.Y. Isseroff R.R. Nuccitelli R. Human keratinocytes migrate to the negative pole in direct current electric fields comparable to those measured in mammalian wounds.J Cell Sci. 1996; 109: 199-207PubMed Google Scholar). The value of cosine equals minus one (cosine = –1) if the cell moves towards the anode; cosine = +1 if the cell moves directly towards the cathode; and is zero if the cell moves perpendicular to the field direction or randomly. For any given condition, a compilation of average cosine from 65 to 150 cells (collected from four to eight experiments) is presented. Also for any given condition, the migration characteristics of control cells exposed to the electric fields in the presence of unmodified KMM are included (Table I and Table II). Statistical analysis was performed by Student t test on all experimental data, using the Instat program.Table IEffects of growth factors on keratinocyte migration in DC electric fieldsReagentsRate (μm per min) (%)aCell migration rate under the indicated conditions is compared in percentage with the rate of control cells.Average cosineCell no. (n)no electric field1.0 ± 0.08 (100)dNot significant at all compared with control of each set.0.06 ± 0.10bp value <0.005 compared with control of each set.119control (+HKGS)1.0 ± 0.05 (100)0.41 ± 0.07102–HKGS0.5 ± 0.03 (50)bp value <0.005 compared with control of each set.0.50 ± 0.06dNot significant at all compared with control of each set.128control1.0 ± 0.06 (100)0.53 ± 0.0871–HKGS + 0.2 ng EGF per ml0.9 ± 0.06 (90)dNot significant at all compared with control of each set.0.62 ± 0.08dNot significant at all compared with control of each set.84–HKGS + 1 ng EGF per ml1.1 ± 0.07 (110)dNot significant at all compared with control of each set.0.64 ± 0.06dNot significant at all compared with control of each set.73–HKGS + 10 ng EGF per ml1.1 ± 0.06 (110)dNot significant at all compared with control of each set.0.61 ± 0.05dNot significant at all compared with control of each set.82control1.0 ± 0.05 (100)0.51 ± 0.0854–HKGS + 5 μg insulin per ml0.7 ± 0.02 (70)bp value <0.005 compared with control of each set.0.47 ± 0.06dNot significant at all compared with control of each set.102–HKGS + 10 μg insulin per ml0.8 ± 0.05 (80)cp value <0.01 compared with control of each set.0.51 ± 0.07dNot significant at all compared with control of each set.70–HKGS + 100 μg insulin per ml0.6 ± 0.03 (60)bp value <0.005 compared with control of each set.0.45 ± 0.07dNot significant at all compared with control of each set.85control0.9 ± 0.04 (100)0.73 ± 0.0580–HKGS + 0.2% vol/vol BPE0.7 ± 0.04 (78)bp value <0.005 compared with control of each set.0.45 ± 0.07cp value <0.01 compared with control of each set.65–HKGS + 2.0% vol/vol BPE0.6 ± 0.03 (67)bp value <0.005 compared with control of each set.0.67 ± 0.05dNot significant at all compared with control of each set.71a Cell migration rate under the indicated conditions is compared in percentage with the rate of control cells.b p value <0.005 compared with control of each set.c p value <0.01 compared with control of each set.d Not significant at all compared with control of each set. Open table in a new tab Table IIEffects of calcium and EGTA on keratinocyte migration in DC electric fieldsReagents (presumed [Ca++])Measured [Ca++]Rate (μm per min) (%)Average cosineCell no. (n)calcium-free (0 mM)2.9 μM0.7 ± 0.03 (86)hp value <0.05 compared with control of each set.–0.43 ± 0.06iNot significant at all compared with control of each set.89KGMaKGM, keratinocyte growth medium; KMM, keratinocyte migration medium. (0.2 mM)0.19 mM0.8 ± 0.03 (100)iNot significant at all compared with control of each set.–0.26 ± 0.06iNot significant at all compared with control of each set.152KMMaKGM, keratinocyte growth medium; KMM, keratinocyte migration medium. (2.0 mM)3.3 mM0.8 ± 0.03 (100)–0.29 ± 0.06136“calcium-free” (0 mM)2.9 μM0.8 ± 0.04 (100)–0.29 ± 0.0779+ 1 mM EGTAcEGTA was added to the “calcium-free” medium immediately before set-up for galvanotaxis, which took about 10–15 min.0.5 ± 0.02 (63)fp value <0.005 compared with control of each set.–0.26 ± 0.07iNot significant at all compared with control of each set.98+ 2 mM EGTAcEGTA was added to the “calcium-free” medium immediately before set-up for galvanotaxis, which took about 10–15 min.0.5 ± 0.02 (63)fp value <0.005 compared with control of each set.–0.25 ± 0.07iNot significant at all compared with control of each set.102+ 4 mM EGTAcEGTA was added to the “calcium-free” medium immediately before set-up for galvanotaxis, which took about 10–15 min.0.3 ± 0.02fp value <0.005 compared with control of each set.–0.00NVbV, not valid. Cells that moved at a rate of 0.3 μm per min or less are considered as no movement.89+ 2 mM EGTA (1 h)dEGTA was added to the “calcium-free” medium 1 h before set-up for galvanotaxis.0.4 ± 0.02 (50)fp value <0.005 compared with control of each set.–0.04 ± 0.08gp value <0.01 compared with control of each set.88eCell migration rate under the indicated conditions is compared in percentage with the rate of control cells.a KGM, keratinocyte growth medium; KMM, keratinocyte migration medium.b V, not valid. Cells that moved at a rate of 0.3 μm per min or less are considered as no movement.c EGTA was added to the “calcium-free” medium immediately before set-up for galvanotaxis, which took about 10–15 min.d EGTA was added to the “calcium-free” medium 1 h before set-up for galvanotaxis.f p value <0.005 compared with control of each set.g p value <0.01 compared with control of each set.h p value <0.05 compared with control of each set.i Not significant at all compared with control of each set. Open table in a new tab eCell migration rate under the indicated conditions is compared in percentage with the rate of control cells. In KMM, human keratinocytes migrated at an average rate of 1.0 ± 0.05 μm per min (ranging from 0.8 to 1.1 μm per min) and migrated towards the cathode with an average cosine of 0.41 ± 0.07 (ranging from 0.30 to 0.55, n = 102) over 1 h exposure to a physiologic electric field of 100 mV per mm (Table 1, Figure 1a). The track cosine, an indicator of the directional movement at a given time interval (every 10 min in our case), showed that cells were less cathodally directed within the first 10–20 min of the field exposure, with cosine averaging 0.1–0.2 (Figure 1b, open bars). Keratinocytes became more cathodally directed thereafter, suggesting that cells need a latent time period to sense and respond to the field. The net cosine , measured as an average cosine from time zero to the indicated time point, increased steadily over the 1 h course of exposure (Figure 1b, hatched bars). On the other hand, the rate of cell migration remained unchanged throughout the entire 1 h exposure, 0.9–1.0 μm per min (data not shown). In the absence of electric fields, human keratinocytes migrated randomly (cosine = 0.06 ± 0.10), yet migrated at the same rate as cells exposed to the fields (1.0 ± 0.08 μm per min, n =119, Table 1, Figure 1a). Data collected from 1 h field exposures are consistent with previous work from our laboratory in which human keratinocytes were exposed to the fields for 2–2.5 h (Nishimura et al., 1996Nishimura K.Y. Isseroff R.R. Nuccitelli R. Human keratinocytes migrate to the negative pole in direct current electric fields comparable to those measured in mammalian wounds.J Cell Sci. 1996; 109: 199-207PubMed Google Scholar;Sheridan et al., 1996Sheridan D.M. Isseroff R.R. Nuccitelli R. Imposition of a physiologic DC electric field alters the migratory response of human