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. 1997 Dec 1;100(11):2881–2891. doi: 10.1172/JCI119837

Hypoxia increases human keratinocyte motility on connective tissue.

E A O'Toole 1, M P Marinkovich 1, C L Peavey 1, M R Amieva 1, H Furthmayr 1, T A Mustoe 1, D T Woodley 1
PMCID: PMC508495  PMID: 9389755

Abstract

Re-epithelialization of skin wounds depends upon the migration of keratinocytes from the cut margins of the wound and is enhanced when human keratinocytes are covered with occlusive dressings that induce hypoxia. In this study, two independent migration assays were used to compare cellular motility on connective tissue components under normoxic or hypoxic conditions. Human keratinocytes apposed to collagens or fibronectin exhibited increased motility when subjected to hypoxic (0.2 or 2% oxygen) conditions compared with normoxic (9 or 20% oxygen) conditions. When compared with normoxic cells, hypoxic keratinocytes exhibited increased expression and redistribution of the lamellipodia-associated proteins (ezrin, radixin, and moesin). Furthermore, hypoxic keratinocytes demonstrated decreased secretion of laminin-5, a laminin isoform known to inhibit keratinocyte motility. Hypoxia did not alter the number of integrin receptors on the cell surface, but did induce enhanced secretion of the 92-kD type IV collagenase. These data demonstrate that hypoxia promotes human keratinocyte motility on connective tissue. Hypoxia-driven motility is associated with increased expression of lamellipodia proteins, increased expression of collagenase and decreased expression of laminin-5, the locomotion brake for keratinocytes.

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Selected References

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  1. Albrecht-Buehler G. The phagokinetic tracks of 3T3 cells. Cell. 1977 Jun;11(2):395–404. doi: 10.1016/0092-8674(77)90057-5. [DOI] [PubMed] [Google Scholar]
  2. Algrain M., Turunen O., Vaheri A., Louvard D., Arpin M. Ezrin contains cytoskeleton and membrane binding domains accounting for its proposed role as a membrane-cytoskeletal linker. J Cell Biol. 1993 Jan;120(1):129–139. doi: 10.1083/jcb.120.1.129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Alper J. C., Welch E. A., Ginsberg M., Bogaars H., Maguire P. Moist wound healing under a vapor permeable membrane. J Am Acad Dermatol. 1983 Mar;8(3):347–353. doi: 10.1016/s0190-9622(83)70038-1. [DOI] [PubMed] [Google Scholar]
  4. Alvarez O. M., Mertz P. M., Eaglstein W. H. The effect of occlusive dressings on collagen synthesis and re-epithelialization in superficial wounds. J Surg Res. 1983 Aug;35(2):142–148. doi: 10.1016/0022-4804(83)90136-1. [DOI] [PubMed] [Google Scholar]
  5. Andersson M., Ostman A., Westermark B., Heldin C. H. Characterization of the retention motif in the C-terminal part of the long splice form of platelet-derived growth factor A-chain. J Biol Chem. 1994 Jan 14;269(2):926–930. [PubMed] [Google Scholar]
  6. Baker S. E., DiPasquale A. P., Stock E. L., Quaranta V., Fitchmun M., Jones J. C. Morphogenetic effects of soluble laminin-5 on cultured epithelial cells and tissue explants. Exp Cell Res. 1996 Nov 1;228(2):262–270. doi: 10.1006/excr.1996.0325. [DOI] [PubMed] [Google Scholar]
  7. Bandyopadhyay R. S., Phelan M., Faller D. V. Hypoxia induces AP-1-regulated genes and AP-1 transcription factor binding in human endothelial and other cell types. Biochim Biophys Acta. 1995 Oct 17;1264(1):72–78. doi: 10.1016/0167-4781(95)00116-x. [DOI] [PubMed] [Google Scholar]
  8. Boyce S. T., Ham R. G. Calcium-regulated differentiation of normal human epidermal keratinocytes in chemically defined clonal culture and serum-free serial culture. J Invest Dermatol. 1983 Jul;81(1 Suppl):33s–40s. doi: 10.1111/1523-1747.ep12540422. [DOI] [PubMed] [Google Scholar]
  9. Caffee H. H., Gallagher T. J. Experiments on the effects of hyperbaric oxygen on flap survival in the pig. Plast Reconstr Surg. 1988 May;81(5):751–754. doi: 10.1097/00006534-198805000-00017. [DOI] [PubMed] [Google Scholar]
  10. Carter W. G., Ryan M. C., Gahr P. J. Epiligrin, a new cell adhesion ligand for integrin alpha 3 beta 1 in epithelial basement membranes. Cell. 1991 May 17;65(4):599–610. doi: 10.1016/0092-8674(91)90092-d. [DOI] [PubMed] [Google Scholar]
  11. Carter W. G., Wayner E. A., Bouchard T. S., Kaur P. The role of integrins alpha 2 beta 1 and alpha 3 beta 1 in cell-cell and cell-substrate adhesion of human epidermal cells. J Cell Biol. 1990 Apr;110(4):1387–1404. doi: 10.1083/jcb.110.4.1387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cha D., O'Brien P., O'Toole E. A., Woodley D. T., Hudson L. G. Enhanced modulation of keratinocyte motility by transforming growth factor-alpha (TGF-alpha) relative to epidermal growth factor (EGF). J Invest Dermatol. 1996 Apr;106(4):590–597. doi: 10.1111/1523-1747.ep12345083. [DOI] [PubMed] [Google Scholar]
  13. Chen J. D., Kim J. P., Zhang K., Sarret Y., Wynn K. C., Kramer R. H., Woodley D. T. Epidermal growth factor (EGF) promotes human keratinocyte locomotion on collagen by increasing the alpha 2 integrin subunit. Exp Cell Res. 1993 Dec;209(2):216–223. doi: 10.1006/excr.1993.1304. [DOI] [PubMed] [Google Scholar]
  14. Chen J. D., Lapiere J. C., Sauder D. N., Peavey C., Woodley D. T. Interleukin-1 alpha stimulates keratinocyte migration through an epidermal growth factor/transforming growth factor-alpha-independent pathway. J Invest Dermatol. 1995 May;104(5):729–733. doi: 10.1111/1523-1747.ep12606970. [DOI] [PubMed] [Google Scholar]
  15. Clark R. A., Lanigan J. M., DellaPelle P., Manseau E., Dvorak H. F., Colvin R. B. Fibronectin and fibrin provide a provisional matrix for epidermal cell migration during wound reepithelialization. J Invest Dermatol. 1982 Nov;79(5):264–269. doi: 10.1111/1523-1747.ep12500075. [DOI] [PubMed] [Google Scholar]
  16. Conlon K. C., Sclafani L., DiResta G. R., Brennan M. F. Comparison of transcutaneous oximetry and laser Doppler flowmetry as noninvasive predictors of wound healing after excision of extremity soft-tissue sarcomas. Surgery. 1994 Mar;115(3):335–340. [PubMed] [Google Scholar]
  17. Crepaldi T., Gautreau A., Comoglio P. M., Louvard D., Arpin M. Ezrin is an effector of hepatocyte growth factor-mediated migration and morphogenesis in epithelial cells. J Cell Biol. 1997 Jul 28;138(2):423–434. doi: 10.1083/jcb.138.2.423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Dunsmore S. E., Rubin J. S., Kovacs S. O., Chedid M., Parks W. C., Welgus H. G. Mechanisms of hepatocyte growth factor stimulation of keratinocyte metalloproteinase production. J Biol Chem. 1996 Oct 4;271(40):24576–24582. doi: 10.1074/jbc.271.40.24576. [DOI] [PubMed] [Google Scholar]
  19. Eaglstein W. H., Davis S. C., Mehle A. L., Mertz P. M. Optimal use of an occlusive dressing to enhance healing. Effect of delayed application and early removal on wound healing. Arch Dermatol. 1988 Mar;124(3):392–395. [PubMed] [Google Scholar]
  20. Eaglstein W. H. Experiences with biosynthetic dressings. J Am Acad Dermatol. 1985 Feb;12(2 Pt 2):434–440. doi: 10.1016/s0190-9622(85)80006-2. [DOI] [PubMed] [Google Scholar]
  21. Eisinger M., Lee J. S., Hefton J. M., Darzynkiewicz Z., Chiao J. W., de Harven E. Human epidermal cell cultures: growth and differentiation in the absence of differentiation in the absence of dermal components or medium supplements. Proc Natl Acad Sci U S A. 1979 Oct;76(10):5340–5344. doi: 10.1073/pnas.76.10.5340. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Falanga V., McKenzie A., Eaglstein W. H. Heterogeneity in oxygen diffusion around venous ulcers. J Dermatol Surg Oncol. 1991 Apr;17(4):336–339. doi: 10.1111/j.1524-4725.1991.tb01707.x. [DOI] [PubMed] [Google Scholar]
  23. Falanga V. Occlusive wound dressings. Why, when, which? Arch Dermatol. 1988 Jun;124(6):872–877. [PubMed] [Google Scholar]
  24. Falanga V., Qian S. W., Danielpour D., Katz M. H., Roberts A. B., Sporn M. B. Hypoxia upregulates the synthesis of TGF-beta 1 by human dermal fibroblasts. J Invest Dermatol. 1991 Oct;97(4):634–637. doi: 10.1111/1523-1747.ep12483126. [DOI] [PubMed] [Google Scholar]
  25. Franzeck U. K., Talke P., Bernstein E. F., Golbranson F. L., Fronek A. Transcutaneous PO2 measurements in health and peripheral arterial occlusive disease. Surgery. 1982 Feb;91(2):156–163. [PubMed] [Google Scholar]
  26. Hammarlund C., Sundberg T. Hyperbaric oxygen reduced size of chronic leg ulcers: a randomized double-blind study. Plast Reconstr Surg. 1994 Apr;93(4):829–834. [PubMed] [Google Scholar]
  27. Helfman T., Falanga V. Gene expression in low oxygen tension. Am J Med Sci. 1993 Jul;306(1):37–41. doi: 10.1097/00000441-199307000-00010. [DOI] [PubMed] [Google Scholar]
  28. Herrick S. E., Ireland G. W., Simon D., McCollum C. N., Ferguson M. W. Venous ulcer fibroblasts compared with normal fibroblasts show differences in collagen but not fibronectin production under both normal and hypoxic conditions. J Invest Dermatol. 1996 Jan;106(1):187–193. doi: 10.1111/1523-1747.ep12329920. [DOI] [PubMed] [Google Scholar]
  29. Hugo C., Hugo C., Pichler R., Gordon K., Schmidt R., Amieva M., Couser W. G., Furthmayr H., Johnson R. J. The cytoskeletal linking proteins, moesin and radixin, are upregulated by platelet-derived growth factor, but not basic fibroblast growth factor in experimental mesangial proliferative glomerulonephritis. J Clin Invest. 1996 Jun 1;97(11):2499–2508. doi: 10.1172/JCI118697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Hunt T. K. The physiology of wound healing. Ann Emerg Med. 1988 Dec;17(12):1265–1273. doi: 10.1016/s0196-0644(88)80351-2. [DOI] [PubMed] [Google Scholar]
  31. Inoue M., Kratz G., Haegerstrand A., Ståhle-Bäckdahl M. Collagenase expression is rapidly induced in wound-edge keratinocytes after acute injury in human skin, persists during healing, and stops at re-epithelialization. J Invest Dermatol. 1995 Apr;104(4):479–483. doi: 10.1111/1523-1747.ep12605917. [DOI] [PubMed] [Google Scholar]
  32. Ishibashi H., Nakagawa K., Nakashima Y., Sueishi K. Conditioned media of carcinoma cells cultured in hypoxic microenvironment stimulate angiogenesis in vitro; relationship to basic fibroblast growth factor. Virchows Arch. 1995;425(6):561–568. doi: 10.1007/BF00199343. [DOI] [PubMed] [Google Scholar]
  33. Iwasaki T., Chen J. D., Kim J. P., Wynn K. C., Woodley D. T. Dibutyryl cyclic AMP modulates keratinocyte migration without alteration of integrin expression. J Invest Dermatol. 1994 Jun;102(6):891–897. doi: 10.1111/1523-1747.ep12383031. [DOI] [PubMed] [Google Scholar]
  34. Jensen P. J., Rodeck U. Autocrine/paracrine regulation of keratinocyte urokinase plasminogen activator through the TGF-alpha/EGF receptor. J Cell Physiol. 1993 May;155(2):333–339. doi: 10.1002/jcp.1041550214. [DOI] [PubMed] [Google Scholar]
  35. Kim J. P., Zhang K., Chen J. D., Wynn K. C., Kramer R. H., Woodley D. T. Mechanism of human keratinocyte migration on fibronectin: unique roles of RGD site and integrins. J Cell Physiol. 1992 Jun;151(3):443–450. doi: 10.1002/jcp.1041510303. [DOI] [PubMed] [Google Scholar]
  36. Kim J. P., Zhang K., Kramer R. H., Schall T. J., Woodley D. T. Integrin receptors and RGD sequences in human keratinocyte migration: unique anti-migratory function of alpha 3 beta 1 epiligrin receptor. J Invest Dermatol. 1992 May;98(5):764–770. doi: 10.1111/1523-1747.ep12499947. [DOI] [PubMed] [Google Scholar]
  37. Kulonen E., Niinikoski J. Effect of hyperbaric oxygenation on wound healing and experimental granuloma. Acta Physiol Scand. 1968 Jul;73(3):383–384. doi: 10.1111/j.1748-1716.1968.tb04116.x. [DOI] [PubMed] [Google Scholar]
  38. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  39. Mani R., White J. E., Barrett D. F., Weaver P. W. Tissue oxygenation, venous ulcers and fibrin cuffs. J R Soc Med. 1989 Jun;82(6):345–346. doi: 10.1177/014107688908200610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Marinkovich M. P., Lunstrum G. P., Burgeson R. E. The anchoring filament protein kalinin is synthesized and secreted as a high molecular weight precursor. J Biol Chem. 1992 Sep 5;267(25):17900–17906. [PubMed] [Google Scholar]
  41. McCawley L. J., O'Brien P., Hudson L. G. Overexpression of the epidermal growth factor receptor contributes to enhanced ligand-mediated motility in keratinocyte cell lines. Endocrinology. 1997 Jan;138(1):121–127. doi: 10.1210/endo.138.1.4844. [DOI] [PubMed] [Google Scholar]
  42. McLean I. W., Nakane P. K. Periodate-lysine-paraformaldehyde fixative. A new fixation for immunoelectron microscopy. J Histochem Cytochem. 1974 Dec;22(12):1077–1083. doi: 10.1177/22.12.1077. [DOI] [PubMed] [Google Scholar]
  43. Mustoe T. A., Pierce G. F., Thomason A., Gramates P., Sporn M. B., Deuel T. F. Accelerated healing of incisional wounds in rats induced by transforming growth factor-beta. Science. 1987 Sep 11;237(4820):1333–1336. doi: 10.1126/science.2442813. [DOI] [PubMed] [Google Scholar]
  44. Nemeth A. J., Eaglstein W. H., Falanga V. Clinical parameters and transcutaneous oxygen measurements for the prognosis of venous ulcers. J Am Acad Dermatol. 1989 Feb;20(2 Pt 1):186–190. doi: 10.1016/s0190-9622(89)70019-0. [DOI] [PubMed] [Google Scholar]
  45. O'Keefe E. J., Chiu M. L. Stimulation of thymidine incorporation in keratinocytes by insulin, epidermal growth factor, and placental extract: comparison with cell number to assess growth. J Invest Dermatol. 1988 Jan;90(1):2–7. doi: 10.1111/1523-1747.ep12462409. [DOI] [PubMed] [Google Scholar]
  46. O'Keefe E. J., Payne R. E., Jr, Russell N., Woodley D. T. Spreading and enhanced motility of human keratinocytes on fibronectin. J Invest Dermatol. 1985 Aug;85(2):125–130. doi: 10.1111/1523-1747.ep12276531. [DOI] [PubMed] [Google Scholar]
  47. O'Toole E. A., Marinkovich M. P., Hoeffler W. K., Furthmayr H., Woodley D. T. Laminin-5 inhibits human keratinocyte migration. Exp Cell Res. 1997 Jun 15;233(2):330–339. doi: 10.1006/excr.1997.3586. [DOI] [PubMed] [Google Scholar]
  48. Odland G., Ross R. Human wound repair. I. Epidermal regeneration. J Cell Biol. 1968 Oct;39(1):135–151. doi: 10.1083/jcb.39.1.135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Pestonjamasp K., Amieva M. R., Strassel C. P., Nauseef W. M., Furthmayr H., Luna E. J. Moesin, ezrin, and p205 are actin-binding proteins associated with neutrophil plasma membranes. Mol Biol Cell. 1995 Mar;6(3):247–259. doi: 10.1091/mbc.6.3.247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Petersen M. J., Woodley D. T., Stricklin G. P., O'Keefe E. J. Enhanced synthesis of collagenase by human keratinocytes cultured on type I or type IV collagen. J Invest Dermatol. 1990 Mar;94(3):341–346. doi: 10.1111/1523-1747.ep12874471. [DOI] [PubMed] [Google Scholar]
  51. Pilcher B. K., Dumin J. A., Sudbeck B. D., Krane S. M., Welgus H. G., Parks W. C. The activity of collagenase-1 is required for keratinocyte migration on a type I collagen matrix. J Cell Biol. 1997 Jun 16;137(6):1445–1457. doi: 10.1083/jcb.137.6.1445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Roth R. N., Weiss L. D. Hyperbaric oxygen and wound healing. Clin Dermatol. 1994 Jan-Mar;12(1):141–156. doi: 10.1016/0738-081x(94)90265-8. [DOI] [PubMed] [Google Scholar]
  53. Saarialho-Kere U. K., Kovacs S. O., Pentland A. P., Olerud J. E., Welgus H. G., Parks W. C. Cell-matrix interactions modulate interstitial collagenase expression by human keratinocytes actively involved in wound healing. J Clin Invest. 1993 Dec;92(6):2858–2866. doi: 10.1172/JCI116906. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Sarret Y., Woodley D. T., Goldberg G. S., Kronberger A., Wynn K. C. Constitutive synthesis of a 92-kDa keratinocyte-derived type IV collagenase is enhanced by type I collagen and decreased by type IV collagen matrices. J Invest Dermatol. 1992 Dec;99(6):836–841. doi: 10.1111/1523-1747.ep12614800. [DOI] [PubMed] [Google Scholar]
  55. Takeuchi K., Sato N., Kasahara H., Funayama N., Nagafuchi A., Yonemura S., Tsukita S., Tsukita S. Perturbation of cell adhesion and microvilli formation by antisense oligonucleotides to ERM family members. J Cell Biol. 1994 Jun;125(6):1371–1384. doi: 10.1083/jcb.125.6.1371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Varghese M. C., Balin A. K., Carter D. M., Caldwell D. Local environment of chronic wounds under synthetic dressings. Arch Dermatol. 1986 Jan;122(1):52–57. [PubMed] [Google Scholar]
  58. Verrando P., Pisani A., Ortonne J. P. The new basement membrane antigen recognized by the monoclonal antibody GB3 is a large size glycoprotein: modulation of its expression by retinoic acid. Biochim Biophys Acta. 1988 Jul 7;942(1):45–56. doi: 10.1016/0005-2736(88)90273-8. [DOI] [PubMed] [Google Scholar]
  59. WINTER G. D. Formation of the scab and the rate of epithelization of superficial wounds in the skin of the young domestic pig. Nature. 1962 Jan 20;193:293–294. doi: 10.1038/193293a0. [DOI] [PubMed] [Google Scholar]
  60. Wayner E. A., Carter W. G. Identification of multiple cell adhesion receptors for collagen and fibronectin in human fibrosarcoma cells possessing unique alpha and common beta subunits. J Cell Biol. 1987 Oct;105(4):1873–1884. doi: 10.1083/jcb.105.4.1873. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Wayner E. A., Carter W. G., Piotrowicz R. S., Kunicki T. J. The function of multiple extracellular matrix receptors in mediating cell adhesion to extracellular matrix: preparation of monoclonal antibodies to the fibronectin receptor that specifically inhibit cell adhesion to fibronectin and react with platelet glycoproteins Ic-IIa. J Cell Biol. 1988 Nov;107(5):1881–1891. doi: 10.1083/jcb.107.5.1881. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Wayner E. A., Garcia-Pardo A., Humphries M. J., McDonald J. A., Carter W. G. Identification and characterization of the T lymphocyte adhesion receptor for an alternative cell attachment domain (CS-1) in plasma fibronectin. J Cell Biol. 1989 Sep;109(3):1321–1330. doi: 10.1083/jcb.109.3.1321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Woodley D. T., Bachmann P. M., O'Keefe E. J. Laminin inhibits human keratinocyte migration. J Cell Physiol. 1988 Jul;136(1):140–146. doi: 10.1002/jcp.1041360118. [DOI] [PubMed] [Google Scholar]
  64. Woodley D. T., Kalebec T., Banes A. J., Link W., Prunieras M., Liotta L. Adult human keratinocytes migrating over nonviable dermal collagen produce collagenolytic enzymes that degrade type I and type IV collagen. J Invest Dermatol. 1986 Apr;86(4):418–423. doi: 10.1111/1523-1747.ep12285689. [DOI] [PubMed] [Google Scholar]
  65. Woodley D. T., Rao C. N., Hassell J. R., Liotta L. A., Martin G. R., Kleinman H. K. Interactions of basement membrane components. Biochim Biophys Acta. 1983 Dec 27;761(3):278–283. doi: 10.1016/0304-4165(83)90077-6. [DOI] [PubMed] [Google Scholar]
  66. Yamamoto T., Noble N. A., Miller D. E., Border W. A. Sustained expression of TGF-beta 1 underlies development of progressive kidney fibrosis. Kidney Int. 1994 Mar;45(3):916–927. doi: 10.1038/ki.1994.122. [DOI] [PubMed] [Google Scholar]

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