Skip to main content
SpringerLink
Log in

Intracellular free calcium and growth changes in single human keratinocytes in response to vitamin D and five 20-epi-analogues

  • Original Contributions
  • Published:
Archives of Dermatological Research Aims and scope Submit manuscript

Abstract

Vitamin D, 1,25(OH)2D3, decreases proliferation and promotes differentiation of keratinocytes, and other keratinocyte differentiation stimuli have been associated with an early rise in intracellular free calcium, [Ca2+]i. We therefore investigated the effect of 1,25(OH)2D3, its precursor D3 and five 20-epi-analogues (EB1089, KH1060, KH1139, MC1288, MC1301) on growth and [Ca2+]i levels of normal human keratinocytes. Cells were cultured in medium MCDB153 with an extracellular calcium concentration of 70 ΜM or 1 mM. All the analogues were more potent than 1,25(OH)2D3 at inducing the morphological changes of differentiation, but D3 was inactive. At concentrations down to 10−8 M 1,25(OH)2D3, caused significant inhibition of growth, as assessed by counting cells and measurement of thymidine labelling. At 5 days 50% inhibition of growth occurred with 64 nM 1,25(OH)2D3 and 3330 nM D3. All the analogues were more potent than 1,25(OH)2D3, and KH1060 inhibited growth at 10−10 M. In single keratinocytes [Ca2+]i was measured by micro-spectrofluorimetric techniques using the dye fura-2. No immediate rise in [Ca2+]i was observed following addition of 1,25(OH)2D3 or the analogues up to 10−6 M. However 10−7 M 1,25(OH)2D3 or the analogues induced a gradual increase in [Ca2+]i, significant at 4 h (P<0.001), which increased further over 2–3 days. D3 had no effect on [Ca2+]i. Increases in [Ca2+]i following the differentiation stimuli of either 2 mM extracellular calcium or 1,25(OH)2D3 were similar at 48 h, increasing from 100 ± 3 nM (mean ± SEM) in control cells to 150 ± 3 nM with 2 mM calcium and 144 ± 6 nM with 10−7 M 1,25(OH)2D3. The effect of extracellular calcium in raising [Ca2+]i within minutes was more rapid than 1,25(OH)2D3, but in combination the two were not additive.

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. Albers KM, Woodrow Setzer R, Taichman LB (1986) Heterogeneity in the replicating population of cultured human epidermal keratinocytes. Differentiation 31: 134–140

    Google Scholar 

  2. Albers KM, Greif F, Woodrow Setzer R, Taichman LB (1987) Cell-cycle withdrawal in cultured keratinocytes. Differentiation 34: 236–240

    Google Scholar 

  3. Berridge MJ (1987) Inositol lipids and DNA replication. Philos Trans R Soc Lond [Biol] 317: 525–536

    Google Scholar 

  4. Bikle DD, Nemanic MK, Gee E, Elias P (1986) 1,25-dihydroxyvitamin D3 production by human keratinocytes: kinetics and regulation. J Clin Invest 78: 566–577

    Google Scholar 

  5. Bikle DD, Nemanic MK, Whitney JO, Elias PW (1986) Neonatal human foreskin keratinocytes produce 1,25-dihydroxyvitamin D3. Biochemistry 25: 1545–1548

    Google Scholar 

  6. Binderup L, Latini S, Binderup E, Bretting C, Calverley M, Hansen K (1991) 20-epi-Vitamin D3 analogues: a novel class of potent regulators of cell growth and immune responses. Biochem Pharmacol 42: 1569–1575

    Google Scholar 

  7. Bittiner B, Bleehen SS, MacNeil S (1991) 1α,25(OH)2-vitamin D3 increases intracellular calcium in human keratinocytes. Br J Dermatol 124: 230–235

    Google Scholar 

  8. Boyce ST, Ham RG (1983) Calcium-regulated differentiation of normal human epidermal keratinocytes in chemically defined clonal culture and serum-free serial culture. J Invest Dermatol 81: 33s-40s

    Google Scholar 

  9. Clemens TL, Adams JS, Horiuchi N, Gilchrest BA, Cho H, Tsuchiya Y, Matsuo N, Suda T, Holick MF (1983) Interaction of 1,25-dihydroxyvitamin-D3 with keratinocytes and fibroblasts from skin of normal subjects and a subject with vitamin-D-dependent rickets, type II: a model for study of the mode of action of 1,25-dihydroxyvitamin-D3. J Clin Endocrinol Metab 56: 824–830

    Google Scholar 

  10. Dlugosz AA, Yuspa SH (1991) Staurosporine induces protein kinase C agonist effects and maturation of normal and neoplastic mouse keratinocytes in vitro. Cancer Res 51: 4677–4684

    Google Scholar 

  11. Downs TR, Wilfinger WW (1983) Fluorometric quantification of DNA in cells and tissues. Anal Biochem 131: 538–547

    Google Scholar 

  12. Hashimoto K, Matsumoto K, Higashiyama M, Nishida Y, Yoshikawa K (1990) Growth-inhibitory effects of 1,25-dihydroxyvitamin D3 on normal and psoriatic keratinocytes. Br J Dermatol 123: 93–98

    Google Scholar 

  13. Hennings H, Holbrock KA (1983) Calcium regulation of cell-cell contact and differentiation of epidermal cells in culture. An ultrastructural survey. Exp Cell Res 143: 127–142

    Google Scholar 

  14. Hennings H, Kruszewski FH, Yuspa SH, Tucker RW (1989) Intracellular calcium alterations in response to increased external calcium in normal and neoplastic keratinocytes. Carcinogenesis 10: 777–780

    Google Scholar 

  15. Jaken S, Yuspa SH (1988) Early signals for keratinocyte differentiation: role of Ca2+-mediated inositol lipid metabolism in normal and neoplastic epidermal cells. Carcinogenesis 9: 1033–1038

    Google Scholar 

  16. Kragballe K (1989) Treatment of psoriasis by the topical application of the novel cholecalciferol analogue calcipotriol (MC903). Arch Dermatol 125: 1647–1652

    Google Scholar 

  17. MacLaughlin JA, Cantley LC, Holick MF (1990) 1,25(OH)2D3 increases calcium and phosphatidylinositol metabolism in differentiating cultured human keratinocytes. J Nutr Biochem 1: 81–87

    Google Scholar 

  18. Matsumoto K, Hashimoto K, Nishida Y, Hashiro M, Yoshikawa K (1990) Growth-inhibitory effects of 1,25-dihydroxyvitamin D3 on normal human keratinocytes cultured in serum-free medium. Biochem Biophys Res Commun 166: 916–923

    Google Scholar 

  19. McLane JA, Katz M, Abdelkader N (1990) Effect of 1,25-dihydroxyvitamin-D3 on human keratinocytes grown under different culture conditions. In Vitro Cell Dev Biol 26: 379–387

    Google Scholar 

  20. Milde P, Hauser U, Simon T, Mall G, Ernst V, Haussler MR, Frosch P, Rauterberg EW (1991) Expression of 1,25-dihydroxyvitamin D3 receptors in normal and psoriatic skin. J Invest Dermatol 97: 230–239

    Google Scholar 

  21. Minghetti PP, Norman AW (1988) 1,25(OH)2-vitamin D3 receptor gene regulation and genetic circuity. FASEB J 2: 3043–3053

    Google Scholar 

  22. Morimoto S, Yoshikawa K (1989) Psoriasis and vitamin D3. Arch Dermatol 125: 231–234

    Google Scholar 

  23. Pillai S, Bikle DD (1991) Role of intracellular-free calcium in the cornified envelope formation of keratinocytes: differences in the mode of action of extracellular calcium and 1,25-dihydroxyvitamin D3. J Cell Physiol 146: 94–100

    Google Scholar 

  24. Pillai S, Bikle DD, Elias PM (1988) 1,25-dihydroxyvitamin D production and receptor binding in human keratinocytes varies with differentiation. J Biol Chem 263: 5390–5395

    Google Scholar 

  25. Pillai S, Bikle DD, Elias PM (1988) Vitamin D and epidermal differentiation: evidence for a role of endogenously produced vitamin D metabolites in keratinocyte differentiation. Skin Pharmacol 1: 149–160

    Google Scholar 

  26. Pillai S, Bikle DD, Mancianti ML, Cline P, Hincenbergs M (1990) Calcium regulation and differentiation of normal human keratinocytes: modulation of differentiation competence by stages of growth and extracellular calcium. J Cell Physiol 143: 294–302

    Google Scholar 

  27. Pittelkow MR, Scott RE (1986) New techniques for the in vitro culture of human skin keratinocytes and perspectives on their use for grafting of patients with extensive burns. Mayo Clin Proc 61: 771–777

    Google Scholar 

  28. Regnier M, Darmon M (1991) 1,25-dihydroxyvitamin D3 stimulates specifically the last steps of epidermal differentiation of cultured human keratinocytes. Differentiation 47: 173–188

    Google Scholar 

  29. Sharpe GR, Fisher C (1991) Time-dependent inhibition of growth of human keratinocytes and fibroblasts by cyclosporin A: effect on keratinocytes at therapeutic blood levels. Br J Dermatol 123: 207–213

    Google Scholar 

  30. Sharpe GR, Gillespie JI, Greenwell JR (1989) An increase in intracellular free calcium is an early event during differentiation of cultured human keratinocytes. FEBS Lett 254: 25–28

    Google Scholar 

  31. Sharpe GR, Fisher C, Gillespie JI, Greenwell JR (1993) Growth and differentiation stimuli induce different and distinct increases in intracellular free calcium in human keratinocytes. Arch Dermatol Res 284: 445–450

    Google Scholar 

  32. Smith EL, Walworth NC, Holick MF (1986) Effects of 1,25-dihydroxyvitamin D3 on the morphologic and biochemical differentiation of cultured human epidermal keratinocytes grown in serum-free conditions. J Invest Dermatol 86: 707–714

    Google Scholar 

  33. Tang W, Ziboh VA, Isseroff R, Martinez D (1987) Novel regulatory action of 1,25-dihydroxyvitamin D on the metabolism of polyphosphoinositides in murine epidermal kertinocytes. J Cell Physiol 132: 131–136

    Google Scholar 

  34. Wille JJ, Pittelkow MR, Shipley GD, Scott RE (1984) Integrated control of growth differentiation of normal human prokeratinocytes cultured on serum-free medium: clonal analyses, growth kinetics, and cell cycle studies. J Cell Physiol 121: 31–44

    Google Scholar 

  35. Yada Y, Ozeki T, Meguro S, Mori S, Nozawa Y (1989) Signal transduction in the onset of terminal keratinocyte differentiation induced by 1α,25-dihydroxyvitamin D3: role of protein kinase C translocation. Biochem Biophys Res Commun 163: 1517–1522

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. University Department of Dermatology, Royal Liverpool University Hospital, P.O. Box 147, L69 3BX, Liverpool, UK

    K. T. Jones & G. R. Sharpe

Authors
  1. K. T. Jones
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. G. R. Sharpe
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Jones, K.T., Sharpe, G.R. Intracellular free calcium and growth changes in single human keratinocytes in response to vitamin D and five 20-epi-analogues. Arch Dermatol Res 286, 123–129 (1994). https://doi.org/10.1007/BF00370738

Download citation

  • Received:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00370738

Key words

Search

Navigation