Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1993 Nov 2;123(4):909–919. doi: 10.1083/jcb.123.4.909

Commitment to differentiation and expression of early differentiation markers in murine keratinocytes in vitro are regulated independently of extracellular calcium concentrations

PMCID: PMC2200160  PMID: 7693721

Abstract

In the epidermis, one of the earliest characterized events in keratinocyte differentiation is the coordinate induction of a pair of keratins specifically expressed in suprabasal cells, keratin 1 (K1) and keratin 10 (K10). Both in vivo and in vitro, extracellular calcium is necessary for several biochemical and structural changes during keratinocyte differentiation. However, it has been unclear if calcium serves as a differentiation signal in keratinocytes. In these studies, expression of suprabasal keratin mRNA and protein is used to test whether the initial differentiation of primary mouse keratinocytes in vitro is dependent on changes in the concentration of extracellular calcium. K1 mRNA was expressed at low levels in cultures of keratinocytes growing on plastic in 0.05 mM calcium but in attached cells was not further induced by increases in the concentration of extracellular calcium. Suspension of the keratinocytes into semi-solid medium induced a rapid and substantial increase in both expression of K1 mRNA and in the percentage of cells expressing suprabasal keratin proteins. The induction was unaffected by the concentration of calcium in the semi-solid medium and could not be enhanced by exposing attached cells to higher calcium before suspension. The induction of K1 mRNA could be inhibited by exposure of the keratinocytes to either EGF or fibronectin. These results suggest that commitment of mouse keratinocytes to terminal differentiation is independent of extracellular calcium and may be regulated primarily by extracellular factors other than calcium.

Full Text

The Full Text of this article is available as a PDF (3.2 MB).

Selected References

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

  1. Adams J. C., Watt F. M. Changes in keratinocyte adhesion during terminal differentiation: reduction in fibronectin binding precedes alpha 5 beta 1 integrin loss from the cell surface. Cell. 1990 Oct 19;63(2):425–435. doi: 10.1016/0092-8674(90)90175-e. [DOI] [PubMed] [Google Scholar]
  2. Adams J. C., Watt F. M. Fibronectin inhibits the terminal differentiation of human keratinocytes. Nature. 1989 Jul 27;340(6231):307–309. doi: 10.1038/340307a0. [DOI] [PubMed] [Google Scholar]
  3. Aneskievich B. J., Fuchs E. Terminal differentiation in keratinocytes involves positive as well as negative regulation by retinoic acid receptors and retinoid X receptors at retinoid response elements. Mol Cell Biol. 1992 Nov;12(11):4862–4871. doi: 10.1128/mcb.12.11.4862. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Choi Y., Fuchs E. TGF-beta and retinoic acid: regulators of growth and modifiers of differentiation in human epidermal cells. Cell Regul. 1990 Oct;1(11):791–809. doi: 10.1091/mbc.1.11.791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Danielson P. E., Forss-Petter S., Brow M. A., Calavetta L., Douglass J., Milner R. J., Sutcliffe J. G. p1B15: a cDNA clone of the rat mRNA encoding cyclophilin. DNA. 1988 May;7(4):261–267. doi: 10.1089/dna.1988.7.261. [DOI] [PubMed] [Google Scholar]
  7. Dlugosz A. A., Yuspa S. H. Coordinate changes in gene expression which mark the spinous to granular cell transition in epidermis are regulated by protein kinase C. J Cell Biol. 1993 Jan;120(1):217–225. doi: 10.1083/jcb.120.1.217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Drozdoff V., Pledger W. J. Cellular response to platelet-derived growth factor (PDGF)-AB after down-regulation of PDGF alpha-receptors. Evidence that functional binding does not require alpha-receptors. J Biol Chem. 1991 Sep 15;266(26):17165–17172. [PubMed] [Google Scholar]
  9. Duden R., Franke W. W. Organization of desmosomal plaque proteins in cells growing at low calcium concentrations. J Cell Biol. 1988 Sep;107(3):1049–1063. doi: 10.1083/jcb.107.3.1049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. Fleckman P., Dale B. A., Holbrook K. A. Profilaggrin, a high-molecular-weight precursor of filaggrin in human epidermis and cultured keratinocytes. J Invest Dermatol. 1985 Dec;85(6):507–512. doi: 10.1111/1523-1747.ep12277306. [DOI] [PubMed] [Google Scholar]
  13. Fuchs E. Epidermal differentiation: the bare essentials. J Cell Biol. 1990 Dec;111(6 Pt 2):2807–2814. doi: 10.1083/jcb.111.6.2807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fuchs E., Green H. Regulation of terminal differentiation of cultured human keratinocytes by vitamin A. Cell. 1981 Sep;25(3):617–625. doi: 10.1016/0092-8674(81)90169-0. [DOI] [PubMed] [Google Scholar]
  15. Gigi-Leitner O., Geiger B., Levy R., Czernobilsky B. Cytokeratin expression in squamous metaplasia of the human uterine cervix. Differentiation. 1986;31(3):191–205. doi: 10.1111/j.1432-0436.1986.tb00400.x. [DOI] [PubMed] [Google Scholar]
  16. Glick A. B., Danielpour D., Morgan D., Sporn M. B., Yuspa S. H. Induction and autocrine receptor binding of transforming growth factor-beta 2 during terminal differentiation of primary mouse keratinocytes. Mol Endocrinol. 1990 Jan;4(1):46–52. doi: 10.1210/mend-4-1-46. [DOI] [PubMed] [Google Scholar]
  17. Green H. Terminal differentiation of cultured human epidermal cells. Cell. 1977 Jun;11(2):405–416. doi: 10.1016/0092-8674(77)90058-7. [DOI] [PubMed] [Google Scholar]
  18. Greenberg C. S., Birckbichler P. J., Rice R. H. Transglutaminases: multifunctional cross-linking enzymes that stabilize tissues. FASEB J. 1991 Dec;5(15):3071–3077. doi: 10.1096/fasebj.5.15.1683845. [DOI] [PubMed] [Google Scholar]
  19. Harding C. R., Scott I. R. Histidine-rich proteins (filaggrins): structural and functional heterogeneity during epidermal differentiation. J Mol Biol. 1983 Nov 5;170(3):651–673. doi: 10.1016/s0022-2836(83)80126-0. [DOI] [PubMed] [Google Scholar]
  20. Hennings H., Michael D., Cheng C., Steinert P., Holbrook K., Yuspa S. H. Calcium regulation of growth and differentiation of mouse epidermal cells in culture. Cell. 1980 Jan;19(1):245–254. doi: 10.1016/0092-8674(80)90406-7. [DOI] [PubMed] [Google Scholar]
  21. Huff C. A., Yuspa S. H., Rosenthal D. Identification of control elements 3' to the human keratin 1 gene that regulate cell type and differentiation-specific expression. J Biol Chem. 1993 Jan 5;268(1):377–384. [PubMed] [Google Scholar]
  22. Jaken S., Yuspa S. H. Early signals for keratinocyte differentiation: role of Ca2+-mediated inositol lipid metabolism in normal and neoplastic epidermal cells. Carcinogenesis. 1988 Jun;9(6):1033–1038. doi: 10.1093/carcin/9.6.1033. [DOI] [PubMed] [Google Scholar]
  23. Kopan R., Traska G., Fuchs E. Retinoids as important regulators of terminal differentiation: examining keratin expression in individual epidermal cells at various stages of keratinization. J Cell Biol. 1987 Jul;105(1):427–440. doi: 10.1083/jcb.105.1.427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Ku W. W., Bernstein I. A. Lectin binding as a probe of proliferative and differentiative phases in primary monolayer cultures of cutaneous keratinocytes. Exp Cell Res. 1988 Apr;175(2):298–316. doi: 10.1016/0014-4827(88)90194-2. [DOI] [PubMed] [Google Scholar]
  25. Marchese C., Rubin J., Ron D., Faggioni A., Torrisi M. R., Messina A., Frati L., Aaronson S. A. Human keratinocyte growth factor activity on proliferation and differentiation of human keratinocytes: differentiation response distinguishes KGF from EGF family. J Cell Physiol. 1990 Aug;144(2):326–332. doi: 10.1002/jcp.1041440219. [DOI] [PubMed] [Google Scholar]
  26. Menon G. K., Elias P. M. Ultrastructural localization of calcium in psoriatic and normal human epidermis. Arch Dermatol. 1991 Jan;127(1):57–63. [PubMed] [Google Scholar]
  27. Molloy C. J., Laskin J. D. Specific alterations in keratin biosynthesis in mouse epidermis in vivo and in explant culture following a single exposure to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate. Cancer Res. 1987 Sep 1;47(17):4674–4680. [PubMed] [Google Scholar]
  28. Olson J. E., Winston J. T., Whitlock J. A., Pledger W. J. Cell cycle-dependent gene expression in V point-arrested BALB/c-3T3 cells. J Cell Physiol. 1993 Feb;154(2):333–342. doi: 10.1002/jcp.1041540217. [DOI] [PubMed] [Google Scholar]
  29. Pillai S., Bikle D. D., Mancianti M. L., Cline P., Hincenbergs M. Calcium regulation of growth and differentiation of normal human keratinocytes: modulation of differentiation competence by stages of growth and extracellular calcium. J Cell Physiol. 1990 May;143(2):294–302. doi: 10.1002/jcp.1041430213. [DOI] [PubMed] [Google Scholar]
  30. Pittelkow M. R., Wille J. J., Jr, Scott R. E. Two functionally distinct classes of growth arrest states in human prokeratinocytes that regulate clonogenic potential. J Invest Dermatol. 1986 Apr;86(4):410–417. doi: 10.1111/1523-1747.ep12285684. [DOI] [PubMed] [Google Scholar]
  31. Rice R. H., Chakravarty R., Chen J., O'Callahan W., Rubin A. L. Keratinocyte transglutaminase: regulation and release. Adv Exp Med Biol. 1988;231:51–61. doi: 10.1007/978-1-4684-9042-8_4. [DOI] [PubMed] [Google Scholar]
  32. 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. 1979 Nov;18(3):681–694. doi: 10.1016/0092-8674(79)90123-5. [DOI] [PubMed] [Google Scholar]
  33. Roop D. R., Hawley-Nelson P., Cheng C. K., Yuspa S. H. Keratin gene expression in mouse epidermis and cultured epidermal cells. Proc Natl Acad Sci U S A. 1983 Feb;80(3):716–720. doi: 10.1073/pnas.80.3.716. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Roop D. R., Huitfeldt H., Kilkenny A., Yuspa S. H. Regulated expression of differentiation-associated keratins in cultured epidermal cells detected by monospecific antibodies to unique peptides of mouse epidermal keratins. Differentiation. 1987;35(2):143–150. doi: 10.1111/j.1432-0436.1987.tb00162.x. [DOI] [PubMed] [Google Scholar]
  35. Roop D. R., Krieg T. M., Mehrel T., Cheng C. K., Yuspa S. H. Transcriptional control of high molecular weight keratin gene expression in multistage mouse skin carcinogenesis. Cancer Res. 1988 Jun 1;48(11):3245–3252. [PubMed] [Google Scholar]
  36. Schweizer J., Winter H. Keratin biosynthesis in normal mouse epithelia and in squamous cell carcinomas. mRNA-dependent alterations of the primary structure of distinct keratin subunits in tumors. J Biol Chem. 1983 Nov 10;258(21):13268–13272. [PubMed] [Google Scholar]
  37. Shipley G. D., Pittelkow M. R. Control of growth and differentiation in vitro of human keratinocytes cultured in serum-free medium. Arch Dermatol. 1987 Nov;123(11):1541a–1544a. [PubMed] [Google Scholar]
  38. Sun T. T., Green H. Differentiation of the epidermal keratinocyte in cell culture: formation of the cornified envelope. Cell. 1976 Dec;9(4 Pt 1):511–521. doi: 10.1016/0092-8674(76)90033-7. [DOI] [PubMed] [Google Scholar]
  39. Toftgard R., Yuspa S. H., Roop D. R. Keratin gene expression in mouse skin tumors and in mouse skin treated with 12-O-tetradecanoylphorbol-13-acetate. Cancer Res. 1985 Nov;45(11 Pt 2):5845–5850. [PubMed] [Google Scholar]
  40. Tsao M. C., Walthall B. J., Ham R. G. Clonal growth of normal human epidermal keratinocytes in a defined medium. J Cell Physiol. 1982 Feb;110(2):219–229. doi: 10.1002/jcp.1041100217. [DOI] [PubMed] [Google Scholar]
  41. Turksen K., Choi Y., Fuchs E. Transforming growth factor alpha induces collagen degradation and cell migration in differentiating human epidermal raft cultures. Cell Regul. 1991 Aug;2(8):613–625. doi: 10.1091/mbc.2.8.613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Watt F. M., Green H. Involucrin synthesis is correlated with cell size in human epidermal cultures. J Cell Biol. 1981 Sep;90(3):738–742. doi: 10.1083/jcb.90.3.738. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Watt F. M., Green H. Stratification and terminal differentiation of cultured epidermal cells. Nature. 1982 Feb 4;295(5848):434–436. doi: 10.1038/295434a0. [DOI] [PubMed] [Google Scholar]
  44. Watt F. M., Hudson D. L., Lamb A. G., Bolsover S. R., Silver R. A., Aitchison M. J., Whitaker M. Mitogens induce calcium transients in both dividing and terminally differentiating keratinocytes. J Cell Sci. 1991 Jun;99(Pt 2):397–405. doi: 10.1242/jcs.99.2.397. [DOI] [PubMed] [Google Scholar]
  45. Watt F. M. Involucrin and other markers of keratinocyte terminal differentiation. J Invest Dermatol. 1983 Jul;81(1 Suppl):100s–103s. doi: 10.1111/1523-1747.ep12540786. [DOI] [PubMed] [Google Scholar]
  46. Watt F. M., Jordan P. W., O'Neill C. H. Cell shape controls terminal differentiation of human epidermal keratinocytes. Proc Natl Acad Sci U S A. 1988 Aug;85(15):5576–5580. doi: 10.1073/pnas.85.15.5576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Watt F. M. Terminal differentiation of epidermal keratinocytes. Curr Opin Cell Biol. 1989 Dec;1(6):1107–1115. doi: 10.1016/s0955-0674(89)80058-4. [DOI] [PubMed] [Google Scholar]
  48. Watt G. A surgical care study. NATNEWS. 1984 Feb;21(2):16–21. [PubMed] [Google Scholar]
  49. Wilke M. S., Hsu B. M., Wille J. J., Jr, Pittelkow M. R., Scott R. E. Biologic mechanisms for the regulation of normal human keratinocyte proliferation and differentiation. Am J Pathol. 1988 Apr;131(1):171–181. [PMC free article] [PubMed] [Google Scholar]
  50. Wille J. J., Jr, Pittelkow M. R., Shipley G. D., Scott R. E. Integrated control of growth and differentiation of normal human prokeratinocytes cultured in serum-free medium: clonal analyses, growth kinetics, and cell cycle studies. J Cell Physiol. 1984 Oct;121(1):31–44. doi: 10.1002/jcp.1041210106. [DOI] [PubMed] [Google Scholar]
  51. Yuspa S. H., Harris C. C. Altered differentiation of mouse epidermal cells treated with retinyl acetate in vitro. Exp Cell Res. 1974 May;86(1):95–105. doi: 10.1016/0014-4827(74)90653-3. [DOI] [PubMed] [Google Scholar]
  52. Yuspa S. H., Kilkenny A. E., Steinert P. M., Roop D. R. Expression of murine epidermal differentiation markers is tightly regulated by restricted extracellular calcium concentrations in vitro. J Cell Biol. 1989 Sep;109(3):1207–1217. doi: 10.1083/jcb.109.3.1207. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES