Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1990 Apr 1;110(4):1149–1168. doi: 10.1083/jcb.110.4.1149

Differential extraction of keratin subunits and filaments from normal human epidermis

PMCID: PMC2116084  PMID: 1691188

Abstract

We have investigated keratin interactions in vivo by sequentially extracting water-insoluble proteins from normal human epidermis with increasing concentrations of urea (2, 4, 6, and 9.5 M) and examining each extract by one- and two-dimensional gel electrophoresis, immunoblot analysis using monoclonal anti-keratin antibodies, and EM. The viable layers of normal human epidermis contain keratins K1, K2, K5, K10/11, K14, and K15, which are sequentially expressed during the course of epidermal differentiation. Only keratins K5, K14, and K15, which are synthesized by epidermal basal cells, were solubilized in 2 M urea. Extraction of keratins K1, K2, and K10/11, which are expressed only in differentiating suprabasal cells, required 4-6 M urea. Negative staining of the 2-M urea extract revealed predominantly keratin filament subunits, whereas abundant intermediate-sized filaments were observed in the 4-urea and 6-M urea extracts. These results indicate that in normal human epidermis, keratins K5, K14, and K15 are more soluble than the differentiation-specific keratins K1, K2, and K10/11. This finding suggests that native keratin filaments of different polypeptide composition have differing properties, despite their similar morphology. Furthermore, the observation of stable filaments in 4 and 6 M urea suggests that epidermal keratins K1, K2, and K10/11, which ultimately form the bulk of the protective, nonviable stratum corneum, may comprise filaments that are unusually resistant to denaturation.

Full Text

The Full Text of this article is available as a PDF (3.8 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Blessing M., Jorcano J. L., Franke W. W. Enhancer elements directing cell-type-specific expression of cytokeratin genes and changes of the epithelial cytoskeleton by transfections of hybrid cytokeratin genes. EMBO J. 1989 Jan;8(1):117–126. doi: 10.1002/j.1460-2075.1989.tb03355.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bowden P. E., Quinlan R. A., Breitkreutz D., Fusenig N. E. Proteolytic modification of acidic and basic keratins during terminal differentiation of mouse and human epidermis. Eur J Biochem. 1984 Jul 2;142(1):29–36. doi: 10.1111/j.1432-1033.1984.tb08246.x. [DOI] [PubMed] [Google Scholar]
  3. Celis J. E., Fey S. J., Larsen P. M., Celis A. Preferential phosphorylation of keratins and vimentin during mitosis in normal and transformed human amnion cells. Ann N Y Acad Sci. 1985;455:268–281. doi: 10.1111/j.1749-6632.1985.tb50417.x. [DOI] [PubMed] [Google Scholar]
  4. Eckert B. S., Caputi S. E., Warren R. H. Dynamics of keratin filaments and the intermediate filament distribution center during shape change in PtK1 cells. Cell Motil. 1984;4(3):169–181. doi: 10.1002/cm.970040303. [DOI] [PubMed] [Google Scholar]
  5. Eichner R., Bonitz P., Sun T. T. Classification of epidermal keratins according to their immunoreactivity, isoelectric point, and mode of expression. J Cell Biol. 1984 Apr;98(4):1388–1396. doi: 10.1083/jcb.98.4.1388. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Eichner R., Rew P., Engel A., Aebi U. Human epidermal keratin filaments: studies on their structure and assembly. Ann N Y Acad Sci. 1985;455:381–402. doi: 10.1111/j.1749-6632.1985.tb50424.x. [DOI] [PubMed] [Google Scholar]
  7. Eichner R., Sun T. T., Aebi U. The role of keratin subfamilies and keratin pairs in the formation of human epidermal intermediate filaments. J Cell Biol. 1986 May;102(5):1767–1777. doi: 10.1083/jcb.102.5.1767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Franke W. W., Schiller D. L., Hatzfeld M., Winter S. Protein complexes of intermediate-sized filaments: melting of cytokeratin complexes in urea reveals different polypeptide separation characteristics. Proc Natl Acad Sci U S A. 1983 Dec;80(23):7113–7117. doi: 10.1073/pnas.80.23.7113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Franke W. W., Schmid E., Grund C., Geiger B. Intermediate filament proteins in nonfilamentous structures: transient disintegration and inclusion of subunit proteins in granular aggregates. Cell. 1982 Aug;30(1):103–113. doi: 10.1016/0092-8674(82)90016-2. [DOI] [PubMed] [Google Scholar]
  10. Franke W. W., Schmid E., Mittnacht S., Grund C., Jorcano J. L. Integration of different keratins into the same filament system after microinjection of mRNA for epidermal keratins into kidney epithelial cells. Cell. 1984 Apr;36(4):813–825. doi: 10.1016/0092-8674(84)90031-x. [DOI] [PubMed] [Google Scholar]
  11. Franke W. W., Weber K., Osborn M., Schmid E., Freudenstein C. Antibody to prekeratin. Decoration of tonofilament like arrays in various cells of epithelial character. Exp Cell Res. 1978 Oct 15;116(2):429–445. doi: 10.1016/0014-4827(78)90466-4. [DOI] [PubMed] [Google Scholar]
  12. Fuchs E. V., Coppock S. M., Green H., Cleveland D. W. Two distinct classes of keratin genes and their evolutionary significance. Cell. 1981 Nov;27(1 Pt 2):75–84. doi: 10.1016/0092-8674(81)90362-7. [DOI] [PubMed] [Google Scholar]
  13. Fuchs E., Green H. Changes in keratin gene expression during terminal differentiation of the keratinocyte. Cell. 1980 Apr;19(4):1033–1042. doi: 10.1016/0092-8674(80)90094-x. [DOI] [PubMed] [Google Scholar]
  14. Giudice G. J., Fuchs E. The transfection of epidermal keratin genes into fibroblasts and simple epithelial cells: evidence for inducing a type I keratin by a type II gene. Cell. 1987 Feb 13;48(3):453–463. doi: 10.1016/0092-8674(87)90196-6. [DOI] [PubMed] [Google Scholar]
  15. Hatzfeld M., Franke W. W. Pair formation and promiscuity of cytokeratins: formation in vitro of heterotypic complexes and intermediate-sized filaments by homologous and heterologous recombinations of purified polypeptides. J Cell Biol. 1985 Nov;101(5 Pt 1):1826–1841. doi: 10.1083/jcb.101.5.1826. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Heid H. W., Werner E., Franke W. W. The complement of native alpha-keratin polypeptides of hair-forming cells: a subset of eight polypeptides that differ from epithelial cytokeratins. Differentiation. 1986;32(2):101–119. doi: 10.1111/j.1432-0436.1986.tb00562.x. [DOI] [PubMed] [Google Scholar]
  17. Horwitz B., Kupfer H., Eshhar Z., Geiger B. Reorganization of arrays of prekeratin filaments during mitosis. Immunofluorescence microscopy with multiclonal and monoclonal prekeratin antibodies. Exp Cell Res. 1981 Aug;134(2):281–290. doi: 10.1016/0014-4827(81)90427-4. [DOI] [PubMed] [Google Scholar]
  18. Kim K. H., Rheinwald J. G., Fuchs E. V. Tissue specificity of epithelial keratins: differential expression of mRNAs from two multigene families. Mol Cell Biol. 1983 Apr;3(4):495–502. doi: 10.1128/mcb.3.4.495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Knapp L. W., Bunn C. L. The experimental manipulation of keratin expression and organization in epithelial cells and somatic cell hybrids. Curr Top Dev Biol. 1987;22:69–96. doi: 10.1016/s0070-2153(08)60099-x. [DOI] [PubMed] [Google Scholar]
  20. Kopan R., Fuchs E. The use of retinoic acid to probe the relation between hyperproliferation-associated keratins and cell proliferation in normal and malignant epidermal cells. J Cell Biol. 1989 Jul;109(1):295–307. doi: 10.1083/jcb.109.1.295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  22. Lane E. B., Goodman S. L., Trejdosiewicz L. K. Disruption of the keratin filament network during epithelial cell division. EMBO J. 1982;1(11):1365–1372. doi: 10.1002/j.1460-2075.1982.tb01324.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lee L. D., Baden H. P. Organisation of the polypeptide chains in mammalian keratin. Nature. 1976 Nov 25;264(5584):377–379. doi: 10.1038/264377a0. [DOI] [PubMed] [Google Scholar]
  24. Lynch M. H., O'Guin W. M., Hardy C., Mak L., Sun T. T. Acidic and basic hair/nail ("hard") keratins: their colocalization in upper cortical and cuticle cells of the human hair follicle and their relationship to "soft" keratins. J Cell Biol. 1986 Dec;103(6 Pt 2):2593–2606. doi: 10.1083/jcb.103.6.2593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lynley A. M., Dale B. A. The characterization of human epidermal filaggrin. A histidine-rich, keratin filament-aggregating protein. Biochim Biophys Acta. 1983 Apr 14;744(1):28–35. doi: 10.1016/0167-4838(83)90336-9. [DOI] [PubMed] [Google Scholar]
  26. Milstone L. M. Isolation and characterization of two polypeptides that form intermediate filaments in bovine esophageal epithelium. J Cell Biol. 1981 Feb;88(2):317–322. doi: 10.1083/jcb.88.2.317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Moll R., Franke W. W., Schiller D. L., Geiger B., Krepler R. The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells. Cell. 1982 Nov;31(1):11–24. doi: 10.1016/0092-8674(82)90400-7. [DOI] [PubMed] [Google Scholar]
  28. Nelson W. G., Sun T. T. The 50- and 58-kdalton keratin classes as molecular markers for stratified squamous epithelia: cell culture studies. J Cell Biol. 1983 Jul;97(1):244–251. doi: 10.1083/jcb.97.1.244. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. O'Farrell P. Z., Goodman H. M., O'Farrell P. H. High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell. 1977 Dec;12(4):1133–1141. doi: 10.1016/0092-8674(77)90176-3. [DOI] [PubMed] [Google Scholar]
  30. Ottaviano Y., Gerace L. Phosphorylation of the nuclear lamins during interphase and mitosis. J Biol Chem. 1985 Jan 10;260(1):624–632. [PubMed] [Google Scholar]
  31. Quinlan R. A., Cohlberg J. A., Schiller D. L., Hatzfeld M., Franke W. W. Heterotypic tetramer (A2D2) complexes of non-epidermal keratins isolated from cytoskeletons of rat hepatocytes and hepatoma cells. J Mol Biol. 1984 Sep 15;178(2):365–388. doi: 10.1016/0022-2836(84)90149-9. [DOI] [PubMed] [Google Scholar]
  32. Quinlan R. A., Hatzfeld M., Franke W. W., Lustig A., Schulthess T., Engel J. Characterization of dimer subunits of intermediate filament proteins. J Mol Biol. 1986 Nov 20;192(2):337–349. doi: 10.1016/0022-2836(86)90369-4. [DOI] [PubMed] [Google Scholar]
  33. Schiller D. L., Franke W. W., Geiger B. A subfamily of relatively large and basic cytokeratin polypeptides as defined by peptide mapping is represented by one or several polypeptides in epithelial cells. EMBO J. 1982;1(6):761–769. doi: 10.1002/j.1460-2075.1982.tb01243.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Schiller D. L., Franke W. W. Limited proteolysis of cytokeratin a by an endogeneous protease: removal of positively charged terminal sequences. Cell Biol Int Rep. 1983 Jan;7(1):3–3. doi: 10.1016/0309-1651(83)90098-x. [DOI] [PubMed] [Google Scholar]
  35. Skerrow D., Skerrow C. J. Tonofilament differentiation in human epidermis, isolation and polypeptide chain composition of keratinocyte subpopulations. Exp Cell Res. 1983 Jan;143(1):27–35. doi: 10.1016/0014-4827(83)90105-2. [DOI] [PubMed] [Google Scholar]
  36. Steinert P. M., Idler W. W., Zimmerman S. B. Self-assembly of bovine epidermal keratin filaments in vitro. J Mol Biol. 1976 Dec 15;108(3):547–567. doi: 10.1016/s0022-2836(76)80136-2. [DOI] [PubMed] [Google Scholar]
  37. Stromer M. H., Huiatt T. W., Richardson R. L., Robson R. M. Disassembly of synthetic 10-nm desmin filaments from smooth muscle into protofilaments. Eur J Cell Biol. 1981 Aug;25(1):136–143. [PubMed] [Google Scholar]
  38. Sun T. T., Shih C., Green H. Keratin cytoskeletons in epithelial cells of internal organs. Proc Natl Acad Sci U S A. 1979 Jun;76(6):2813–2817. doi: 10.1073/pnas.76.6.2813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sun T. T., Tseng S. C., Huang A. J., Cooper D., Schermer A., Lynch M. H., Weiss R., Eichner R. Monoclonal antibody studies of mammalian epithelial keratins: a review. Ann N Y Acad Sci. 1985;455:307–329. doi: 10.1111/j.1749-6632.1985.tb50419.x. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. Tseng S. C., Jarvinen M. J., Nelson W. G., Huang J. W., Woodcock-Mitchell J., Sun T. T. Correlation of specific keratins with different types of epithelial differentiation: monoclonal antibody studies. Cell. 1982 Sep;30(2):361–372. doi: 10.1016/0092-8674(82)90234-3. [DOI] [PubMed] [Google Scholar]
  42. Tölle H. G., Weber K., Osborn M. Keratin filament disruption in interphase and mitotic cells--how is it induced? Eur J Cell Biol. 1987 Feb;43(1):35–47. [PubMed] [Google Scholar]
  43. Woodcock-Mitchell J., Eichner R., Nelson W. G., Sun T. T. Immunolocalization of keratin polypeptides in human epidermis using monoclonal antibodies. J Cell Biol. 1982 Nov;95(2 Pt 1):580–588. doi: 10.1083/jcb.95.2.580. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES