Skip to main content
SpringerLink
Log in

Differentiation of cultured human keratinocytes: Effect of culture conditions on lipid composition of normal vs. malignant cells

  • Regular Papers
  • Published:
In Vitro Cellular & Developmental Biology Aims and scope Submit manuscript

Summary

Differentiation in keratinocytes can be experimentally modulated by changing the culture conditions. When cultured under conventional, submerged conditions, the extent of cellular differentiation is reduced in the presence of low calcium medium and is enhanced in medium containing physiologic calcium concentrations. Moreover cultures grown at the air-medium interface or on a dermal substrate, or both, differentiate even further. Herein we report the effect of culture conditions on lipid composition in normal human keratinocytes and three squamous carcinoma cell (SCC) lines that vary in their capacity to differentiate as assessed by cornified envelope formation. Under submerged conditions, the total phospholipid content was lower, triglyceride content higher, and phospholipid: neutral lipid ratio lower in direct correlation to the degree of differentiation in these cultures. When grown at the air-medium interface on the-epidermized dermis, evidence of further morphologic differentiation was found only for well-differentiated SCC cells and normal keratinocytes. Similarly, the phospholipid content remained high in poorly differentiated SCC cells and it, decreased modestly in well-differentiated SCC cells and markedly in normal keratinocytes. In all cell lines the triglyceride content was increased and cholesterol content decreased when compared to parallel submerged cultures, but these differences were most pronounced in well-differentiated cell lines. Acylceramides and acylglucosylceramides were found only in normal keratinocytes and only under the most differentiation-enhancing conditions. These studies demonstrate differentiation-related changes in the lipid content of both normal and neoplastic keratinocytes.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Anton-Lamprecht, I. Zur Ultrastruktur hereditarer Verhorungsstorungen. I. Ichthoyosis congenita. Arch Dermatol Forsch 243:88–100; 1972.

    Article  PubMed  CAS  Google Scholar 

  2. Bernstam, L. I.; Vaughn, F. L.; Bernstein, I. A. Keratinocytes grown at the air-liquid interface. In Vitro 22:695–705; 1986.

    CAS  Google Scholar 

  3. Bligh, E. G.; Dyer, W. J. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37:911–917; 1959.

    PubMed  CAS  Google Scholar 

  4. Boonstra, J.; de Laat, S. W.; Ponec, M. Epidermal growth factor receptor expression related to differentiation capacity in normal and transformed keratinocytes. Exp. Cell Res. 161:421–433; 1985.

    Article  PubMed  CAS  Google Scholar 

  5. Boukamp, P.; Rupniak, H. T. R.; Fusenig, N. E. Environmental modulation of the expression of differentiation and malignancy in six human squamous cell carcinoma cell lines. Cancer Res. 45:5582–5592; 1985.

    PubMed  CAS  Google Scholar 

  6. Brody, I. The ultrastructure of the epidermis in psoriasis vulgaris as revealed by electron microscopy. J. Ultrastruct. Res. 6:314–353; 1962.

    Google Scholar 

  7. Brysk, M. M.; Miller, J.; Walker, G. K. Characteristics of a human epidermal squamous carcinoma cell line at different extracellular calcium concentration. Exp. Cell Res. 150:329–337; 1984.

    Article  PubMed  CAS  Google Scholar 

  8. Dale, B. S.; Scofield, J. A. H.; Hennings, H. D., et al. Identification of filaggrin in cultured mouse keratinocytes. J. Invest. Dermatol. 81s:90–95; 1983.

    Article  Google Scholar 

  9. Elias, P. M. Epidermal lipids, barrier function, and desquamation. J. Invest. Dermatol. 80:44–49; 1983.

    Article  CAS  Google Scholar 

  10. Elias, P. M.; Williams, M. L.; Maloney, M. E., et al. Stratum corneum lipids in disorders of cornification: steroid sulfatase and cholesterol sulfate in normal desquamation and the pathogenesis of recessive X-linked ichthyosis. J. Clin. Invest. 74:1414–1421; 1984.

    Article  PubMed  CAS  Google Scholar 

  11. Epstein, E. H., Jr.; Williams, M. L.; Elias, P. M. Steroid sulfatase X-linked ichthyosis and stratum corneum cell cohesion. Arch. Dermatol. 117:761–763; 1984.

    Article  Google Scholar 

  12. Feingold, K. R.; Williams, M. L.; Pillai, S., et al. The effect of vitamin D status on cutaneous sterologenesisin vivo, andin vitro. Biochem. Biophys. Acta 930:193–200; 1987.

    Article  PubMed  CAS  Google Scholar 

  13. Freeman, A. E.; Ingel, V. J.; Herrman, B. J., et al. Growth and characterization of human skin epithelial cell cultures. In Vitro 12:352–362; 1976.

    PubMed  CAS  Google Scholar 

  14. Green, H. Cyclic AMP in relation to proliferation of the epidermal cell: a new view. Cell 15:801–811; 1978.

    Article  PubMed  CAS  Google Scholar 

  15. Green, H.; Kehinde, O.; Thomas, J. Growth of cultured human epidermal cells into multiple epithelial suitable for grafting. Proc. Natl. Acad. Sci. USA 76:5665–5668; 1979.

    Article  PubMed  CAS  Google Scholar 

  16. Hennings H. D.; Michael, D.; Cheng, C., et al. Calcium regulation of growth and differentiation of mouse epidermal cells in culture. Cell 19:245–254; 1980.

    Article  PubMed  CAS  Google Scholar 

  17. Hennings, H. D.; Holbrook, K. A.; Yuspa, S. H. Factors influencing calcium-induced terminal differentiation in cultured mouse epidermal cells. J. cell Physiol. 116:265–281; 1983.

    Article  PubMed  CAS  Google Scholar 

  18. Holbrook, K. A.; Hennings, H. Phenotypic expression of epidermal cells in vitro: a review. J. Invest. Dermatol. 81:11s-24s; 1983.

    Article  PubMed  CAS  Google Scholar 

  19. Kanerva, L.; Lauhasanta, J.; Niemi, K-M., et al. New observations on the fine structure of lamellar ichthyosis and the effect of treatment with etretinate. Am. J. Dermatopathol 5:555–568; 1983.

    Article  PubMed  CAS  Google Scholar 

  20. Kim, K. H.; Schwartz, F.; Fuchs, E. Differences in keratin synthesis between normal epithelial cells and squamous cell carcinoma are mediated by vitamin A. Proc. Natl. Acad. Sci. USA 81:4280–4284; 1985.

    Article  Google Scholar 

  21. Kopan, R.; Traska, G.; Fuchs, E. Retinoids as important regulation of terminal differentiation: examining keratin expression in individual epidermal cells at various stages of keratinization. J. Cell Biol. 105:427–440; 1987.

    Article  PubMed  CAS  Google Scholar 

  22. Lampe, M. A.; Burlingame, A. L.; Whitney, J., et al. Human stratum corneum lipids: characterization and regional variations. J. Lipid Res. 24:120–130; 1983.

    PubMed  CAS  Google Scholar 

  23. Lampe, M. A.; Williams, M. L.; Elias, P. M. Human epidermal lipids: characterization and modulation during differentiation. J. Lipid Res. 24:141–140; 1983.

    Google Scholar 

  24. Lillie, J. H.; MacCallum, D. K.; Jepsen, A. Fine structure of subcultivated stratified squamous epithelial grown on collagen rafts. Cell Res. 125:153–165; 1980.

    Article  CAS  Google Scholar 

  25. Lowry O. H.; Rosebrough, N. J.; Farr, A. L. et al. Protein measurement with the Folin reagent. J. Biol. Chem. 193:265–275; 1951.

    PubMed  CAS  Google Scholar 

  26. Monger, D. J.; Williams, M. L.; Feingold, K. R., et al. Localization of sites of lipid metabolism within murine epidermis. J. Lipid Res. 29:603–612; 1988.

    PubMed  CAS  Google Scholar 

  27. Nemanic, M. K.; Whitney, J.; Elias, P. M.In vitro synthesis of vitamin D3 by cultured human keratinocytes and fibroblasts: action spectrum and effects of AY-9944. Biochim. Biophys. Acta 841:267–277; 1985.

    PubMed  CAS  Google Scholar 

  28. Ponec, M.; Havekes, L.; Kempenaar, J., et al. Defective low-density lipoprotein metabolism in cultured normal, transformed and malignant keratinocytes. J. Invest. Dermatol. 83:436–440; 1984.

    Article  PubMed  CAS  Google Scholar 

  29. Ponec, M.; Havekes, L.; Kempenaar, J.; et al. Calcium-mediated regulation of the low density lipoprotein receptor and intracellular cholesterol synthesis in human epidermal keratinocytes. J. Cell Physiol. 125:98–106; 1985.

    Article  PubMed  CAS  Google Scholar 

  30. Ponec, M.; Kempenaar, J.; Boonstra, J. Regulation of lipid synthesis in relation to keratinocyte differentiation. Biochim. Biophys. Acta 921:512–521; 1987.

    PubMed  CAS  Google Scholar 

  31. Ponec, M.; Kempenaar, J.; van der Schroeff, J. G. Modulating effects of retinoic acid on the morphology of normal human keratinocytes and squamous carcinoma cells cultured on air-liquid interface. J. Invest. Dermatol. 89:310–311; 1987.

    Article  Google Scholar 

  32. Ponec, M.; Weerheim, A.; Kempenaar, J., et al. Lipid composition of cultured human keratinocytes in relation to their differentiation. J. Lipid Res. 29:949–961; 1988.

    PubMed  CAS  Google Scholar 

  33. Rearick, J. I.; Jetten, A. M. Accumulation of cholesterol-3-sulfate during in vitro squamous differentiation of rabbit treated epithelial cells in its regulation by retinoids. J. Biol. Chem. 261:13898–13904; 1986.

    PubMed  CAS  Google Scholar 

  34. Regnier, M.; Prunieras, M.; Woodley, D. Growth and differentiation of adult human epidermal cells on dermal substrate. Front. Matrix Biol. 9:4–35; 1981.

    Google Scholar 

  35. Rheinwald, J. G.; Green, H. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from the single cells. Cell 6:331–344; 1975.

    Article  PubMed  CAS  Google Scholar 

  36. Rheinwald, J. G.; Green, H. Epidermal growth factor and the multiplication of cultured human epidermal keratinocytes. Nature 265:421–424; 1977.

    Article  PubMed  CAS  Google Scholar 

  37. Rheinwald, J. G.; Beckett, M. A. Defective terminal differentiation in cultured as a consistent and selectable character of malignant human keratinocytes. Cell 22:629–632; 1980.

    Article  PubMed  CAS  Google Scholar 

  38. Reinwald, J. G.; Beckett, M. A. Tumorigenic keratinocyte lines requiring anchorage and fibroblast, support cultured from human squamous cell carcinoma. Cancer Res. 41:1657–1663; 1981.

    Google Scholar 

  39. Rice, R. H.; Green H. Presence in human epidermal cells of a soluble protein precursor of the cross-linked envelope: activation of the cross-linking by calcium ions. Cell 18:681–694; 1979.

    Article  PubMed  CAS  Google Scholar 

  40. Stanley, J. R.; Yuspa, S. H. Specific epidermal protein markers are modulated during calcium-induced terminal differentiation. J. Cell Biol. 96:1809–1814; 1983.

    Article  PubMed  CAS  Google Scholar 

  41. Van Muijen, G. N. P.; Warnaar, S. O.; Ponec, M. Differentiation related changes of cytokeratin expression in cultured keratinocytes and in fetal, newborn and adult epidermis. Exp. Cell Res. 171:331–345; 1987.

    Article  PubMed  Google Scholar 

  42. Watt, F. M.; Green, H. Stratification and terminal differentiation of cultured epidermal cells. Nature 295:434–436; 1982.

    Article  PubMed  CAS  Google Scholar 

  43. Watt, F. M.; Mattey, D. L.; Garrod, D. R. Calcium-induced reorganization of desmosomal components in cultured human keratinocytes. J. Cell Biol. 99:2211–2215; 1984.

    Article  PubMed  CAS  Google Scholar 

  44. Wertz, P. W.; Downing, D. T. Glycolipids in mammalian epidermis: structure and function on the water barrier. J. Lipid Res. 24:753–757; 1983.

    PubMed  CAS  Google Scholar 

  45. Wertz, P. W.; Downing, D. T. Glucosylceramides of pig epidermis: structure determination. J. Lipid Res. 24:1135–1139; 1983.

    PubMed  CAS  Google Scholar 

  46. Williams, M. L.; Brown, B. E.; Monger, D. J., et al. Lipid concent and metabolism of human keratinocytes grown at the air-medium interface. J. Cell Physiol. 13:103–110; 1988.

    Article  Google Scholar 

  47. Williams, M. L.; Rutherford, S. L.; Ponec, M., et al. Density-dependent variations in the lipid content and metabolism of cultured human keratinocytes. J. Invest. Dermatol. 91:86–91; 1988.

    Article  PubMed  CAS  Google Scholar 

  48. Yardley, H. J.; Summerly, R. Lipid composition and metabolism in normal and diseased epidermis. Pharmacol & Ther. 13:357–383; 1981.

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Dermatology Service, Veterans Administration Medical Center, San Francisco, California

    Peter M. Elias & Mary L. Williams

  2. Departments of Dermatology and Pediatrics, University of California, San Francisco, California

    Peter M. Elias & Mary L. Williams

  3. Department of Dermatology, University Hospital Leiden, P. O. Box 9600, 2300 RC, Leiden, The Netherlands

    Maria Ponec, Arij Weerheim & Johanna Kempenaar

Authors
  1. Maria Ponec
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Arij Weerheim
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Johanna Kempenaar
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Peter M. Elias
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Mary L. Williams
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

This work was supported in part by NATO Scientific Award (RG 8510056).

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ponec, M., Weerheim, A., Kempenaar, J. et al. Differentiation of cultured human keratinocytes: Effect of culture conditions on lipid composition of normal vs. malignant cells. In Vitro Cell Dev Biol 25, 689–696 (1989). https://doi.org/10.1007/BF02623721

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02623721

Key words

Search

Navigation