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. 1989 Sep 1;109(3):1207–1217. doi: 10.1083/jcb.109.3.1207

Expression of murine epidermal differentiation markers is tightly regulated by restricted extracellular calcium concentrations in vitro

PMCID: PMC2115750  PMID: 2475508

Abstract

Epidermal differentiation is characterized by a series of coordinated morphological and biochemical changes which result in a highly specialized, highly organized, stratified squamous epithelium. Among the specific markers expressed in differentiating epidermis are (a) two early spinous cell proteins, keratins 1 and 10 (K1 and K10); and (b) two later granular cell proteins, filaggrin and a cornified envelope precursor (CE). In vitro, epidermal basal cells are selectively cultured in 0.05 mM Ca2+ medium, and terminal differentiation is induced when the Ca2+ concentration is increased to 1 mM. However, only a small fraction of the cells express the markers K1, K10, CE, or filaggrin in the higher Ca2+ medium. To explore the factors required for marker expression, cultured epidermal cells were exposed to intermediate Ca2+ concentrations and extracts were analyzed using specific antibody and nucleic acid probes for the four markers of interest. These studies revealed that marker expression was enhanced at a restricted concentration of Ca2+ in the medium of 0.10-0.16 mM. At this Ca2+ concentration, both protein and mRNA levels for each marker were substantially increased, whereas at higher or lower Ca2+ concentrations they were diminished or undetected. The percentage of cells expressing each marker was increased two- to threefold in the permissive Ca2+ medium as determined by immunofluorescence analysis. This optimal level of Ca2+ was required both to initiate and sustain marker expression. At the permissive Ca2+ concentration, expression of the markers was sequential and similar to the order of appearance in vivo. K1 was expressed within 8-12 h and K10 was expressed in the ensuing 12-24-h period. CE and filaggrin were expressed in the subsequent 24 h. Inhibition of K1 expression by cycloheximide suggested that an inducible protein was involved. Other investigators have determined that a shallow Ca2+ gradient exists in epidermis, where the basal cells and spinous cells are in a Ca2+ environment substantially below serum Ca2+ levels. These in vitro results suggest that the Ca2+ environment is a fundamental regulator of expression of epidermal differentiation markers and provide an explanation for the existence of the Ca2+ gradient in vivo.

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Selected References

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  1. Alwine J. C., Kemp D. J., Stark G. R. Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5350–5354. doi: 10.1073/pnas.74.12.5350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Asselineau D., Bernhard B., Bailly C., Darmon M. Epidermal morphogenesis and induction of the 67 kD keratin polypeptide by culture of human keratinocytes at the liquid-air interface. Exp Cell Res. 1985 Aug;159(2):536–539. doi: 10.1016/s0014-4827(85)80027-6. [DOI] [PubMed] [Google Scholar]
  3. Berridge M. J. Inositol lipids and DNA replication. Philos Trans R Soc Lond B Biol Sci. 1987 Dec 15;317(1187):525–536. doi: 10.1098/rstb.1987.0079. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Bramhall S., Noack N., Wu M., Loewenberg J. R. A simple colorimetric method for determination of protein. Anal Biochem. 1969 Oct 1;31(1):146–148. doi: 10.1016/0003-2697(69)90251-6. [DOI] [PubMed] [Google Scholar]
  6. Breitkreutz D., Bohnert A., Herzmann E., Bowden P. E., Boukamp P., Fusenig N. E. Differentiation specific functions in cultured and transplanted mouse keratinocytes: environmental influences on ultrastructure and keratin expression. Differentiation. 1984;26(2):154–169. doi: 10.1111/j.1432-0436.1984.tb01389.x. [DOI] [PubMed] [Google Scholar]
  7. Buxman M. M., Buehner G. E., Weupper K. D. Isolation of substrates of epidermal transglutaminase from bovine epidermis. Biochem Biophys Res Commun. 1976 Nov 22;73(2):470–478. doi: 10.1016/0006-291x(76)90731-2. [DOI] [PubMed] [Google Scholar]
  8. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  9. Dale B. A., Holbrook K. A., Steinert P. M. Assembly of stratum corneum basic protein and keratin filaments in macrofibrils. Nature. 1978 Dec 14;276(5689):729–731. doi: 10.1038/276729a0. [DOI] [PubMed] [Google Scholar]
  10. Dover R., Watt F. M. Measurement of the rate of epidermal terminal differentiation: expression of involucrin by S-phase keratinocytes in culture and in psoriatic plaques. J Invest Dermatol. 1987 Oct;89(4):349–352. doi: 10.1111/1523-1747.ep12471751. [DOI] [PubMed] [Google Scholar]
  11. Eady R. A. The basement membrane. Interface between the epithelium and the dermis: structural features. Arch Dermatol. 1988 May;124(5):709–712. doi: 10.1001/archderm.124.5.709. [DOI] [PubMed] [Google Scholar]
  12. Eckert R. L., Green H. Structure and evolution of the human involucrin gene. Cell. 1986 Aug 15;46(4):583–589. doi: 10.1016/0092-8674(86)90884-6. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. 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]
  16. Fuchs E., Green H. Multiple keratins of cultured human epidermal cells are translated from different mRNA molecules. Cell. 1979 Jul;17(3):573–582. doi: 10.1016/0092-8674(79)90265-4. [DOI] [PubMed] [Google Scholar]
  17. Hanukoglu I., Fuchs E. The cDNA sequence of a Type II cytoskeletal keratin reveals constant and variable structural domains among keratins. Cell. 1983 Jul;33(3):915–924. doi: 10.1016/0092-8674(83)90034-x. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Hennings H., Kruszewski F. H., Yuspa S. H., Tucker R. W. Intracellular calcium alterations in response to increased external calcium in normal and neoplastic keratinocytes. Carcinogenesis. 1989 Apr;10(4):777–780. doi: 10.1093/carcin/10.4.777. [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. Huszar M., Gigi-Leitner O., Moll R., Franke W. W., Geiger B. Monoclonal antibodies to various acidic (type I) cytokeratins of stratified epithelia. Selective markers for stratification and squamous cell carcinomas. Differentiation. 1986;31(2):141–153. doi: 10.1111/j.1432-0436.1986.tb00395.x. [DOI] [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. Kubilus J., Kvedar J., Baden H. P. Identification of new components of the cornified envelope of human and bovine epidermis. J Invest Dermatol. 1987 Jul;89(1):44–50. doi: 10.1111/1523-1747.ep12580376. [DOI] [PubMed] [Google Scholar]
  25. Lechner J. F., Haugen A., McClendon I. A., Pettis E. W. Clonal growth of normal adult human bronchial epithelial cells in a serum-free medium. In Vitro. 1982 Jul;18(7):633–642. doi: 10.1007/BF02796396. [DOI] [PubMed] [Google Scholar]
  26. Lichti U., Yuspa S. H. Modulation of tissue and epidermal transglutaminases in mouse epidermal cells after treatment with 12-O-tetradecanoylphorbol-13-acetate and/or retinoic acid in vivo and in culture. Cancer Res. 1988 Jan 1;48(1):74–81. [PubMed] [Google Scholar]
  27. Lonsdale-Eccles J. D., Resing K. A., Meek R. L., Dale B. A. High-molecular-weight precursor of epidermal filaggrin and hypothesis for its tandem repeating structure. Biochemistry. 1984 Mar 13;23(6):1239–1245. doi: 10.1021/bi00301a034. [DOI] [PubMed] [Google Scholar]
  28. McGrath C. M., Soule H. D. Calcium regulation of normal human mammary epithelial cell growth in culture. In Vitro. 1984 Aug;20(8):652–662. doi: 10.1007/BF02619616. [DOI] [PubMed] [Google Scholar]
  29. Menon G. K., Grayson S., Elias P. M. Ionic calcium reservoirs in mammalian epidermis: ultrastructural localization by ion-capture cytochemistry. J Invest Dermatol. 1985 Jun;84(6):508–512. doi: 10.1111/1523-1747.ep12273485. [DOI] [PubMed] [Google Scholar]
  30. Nagae S., Lichti U., De Luca L. M., Yuspa S. H. Effect of retinoic acid on cornified envelope formation: difference between spontaneous envelope formation in vivo or in vitro and expression of envelope competence. J Invest Dermatol. 1987 Jul;89(1):51–58. doi: 10.1111/1523-1747.ep12580383. [DOI] [PubMed] [Google Scholar]
  31. Nemeth E. F., Scarpa A. Rapid mobilization of cellular Ca2+ in bovine parathyroid cells evoked by extracellular divalent cations. Evidence for a cell surface calcium receptor. J Biol Chem. 1987 Apr 15;262(11):5188–5196. [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., Cheng C. K., Titterington L., Meyers C. A., Stanley J. R., Steinert P. M., Yuspa S. H. Synthetic peptides corresponding to keratin subunits elicit highly specific antibodies. J Biol Chem. 1984 Jul 10;259(13):8037–8040. [PubMed] [Google Scholar]
  34. 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]
  35. 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]
  36. 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]
  37. Rothnagel J. A., Mehrel T., Idler W. W., Roop D. R., Steinert P. M. The gene for mouse epidermal filaggrin precursor. Its partial characterization, expression, and sequence of a repeating filaggrin unit. J Biol Chem. 1987 Nov 15;262(32):15643–15648. [PubMed] [Google Scholar]
  38. Régnier M., Vaigot P., Darmon M., Pruniéras M. Onset of epidermal differentiation in rapidly proliferating basal keratinocytes. J Invest Dermatol. 1986 Oct;87(4):472–476. doi: 10.1111/1523-1747.ep12455517. [DOI] [PubMed] [Google Scholar]
  39. Schweizer J., Kinjo M., Fürstenberger G., Winter H. Sequential expression of mRNA-encoded keratin sets in neonatal mouse epidermis: basal cells with properties of terminally differentiating cells. Cell. 1984 May;37(1):159–170. doi: 10.1016/0092-8674(84)90311-8. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. Silver J., Russell J., Sherwood L. M. Regulation by vitamin D metabolites of messenger ribonucleic acid for preproparathyroid hormone in isolated bovine parathyroid cells. Proc Natl Acad Sci U S A. 1985 Jun;82(12):4270–4273. doi: 10.1073/pnas.82.12.4270. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Simon M., Green H. Participation of membrane-associated proteins in the formation of the cross-linked envelope of the keratinocyte. Cell. 1984 Apr;36(4):827–834. doi: 10.1016/0092-8674(84)90032-1. [DOI] [PubMed] [Google Scholar]
  43. Steinert P. M., Cantieri J. S., Teller D. C., Lonsdale-Eccles J. D., Dale B. A. Characterization of a class of cationic proteins that specifically interact with intermediate filaments. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4097–4101. doi: 10.1073/pnas.78.7.4097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Steinert P. M., Roop D. R. Molecular and cellular biology of intermediate filaments. Annu Rev Biochem. 1988;57:593–625. doi: 10.1146/annurev.bi.57.070188.003113. [DOI] [PubMed] [Google Scholar]
  45. Steinert P. M. The dynamic phosphorylation of the human intermediate filament keratin 1 chain. J Biol Chem. 1988 Sep 15;263(26):13333–13339. [PubMed] [Google Scholar]
  46. Tang W., Ziboh V. A., Isseroff R., Martinez D. Turnover of inositol phospholipids in cultured murine keratinocytes: possible involvement of inositol triphosphate in cellular differentiation. J Invest Dermatol. 1988 Jan;90(1):37–43. doi: 10.1111/1523-1747.ep12462536. [DOI] [PubMed] [Google Scholar]
  47. Thacher S. M., Rice R. H. Keratinocyte-specific transglutaminase of cultured human epidermal cells: relation to cross-linked envelope formation and terminal differentiation. Cell. 1985 Mar;40(3):685–695. doi: 10.1016/0092-8674(85)90217-x. [DOI] [PubMed] [Google Scholar]
  48. 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]
  49. Tseng H., Green H. Remodeling of the involucrin gene during primate evolution. Cell. 1988 Aug 12;54(4):491–496. doi: 10.1016/0092-8674(88)90070-0. [DOI] [PubMed] [Google Scholar]
  50. Van Neste D. J., Staquet M. J., Leroy B. P., De Coster W. J. Distribution pattern of psoriatic keratoblasts: computer-assisted image-analysis for combined evaluation of DNA synthesis and expression of 67 kD keratin polypeptides in the epidermis of stable plaques of psoriasis. J Invest Dermatol. 1988 Mar;90(3):382–386. doi: 10.1111/1523-1747.ep12456446. [DOI] [PubMed] [Google Scholar]
  51. Wark J. D., Tashjian A. H., Jr Regulation of prolactin mRNA by 1,25-dihydroxyvitamin D3 in GH4C1 cells. J Biol Chem. 1983 Oct 25;258(20):12118–12121. [PubMed] [Google Scholar]
  52. Wilke M. S., Hsu B. M., Scott R. E. Two subtypes of reversible cell cycle restriction points exist in cultured normal human keratinocyte progenitor cells. Lab Invest. 1988 Jun;58(6):660–666. [PubMed] [Google Scholar]
  53. 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]
  54. Yuspa S. H., Ben T., Hennings H., Lichti U. Divergent responses in epidermal basal cells exposed to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate. Cancer Res. 1982 Jun;42(6):2344–2349. [PubMed] [Google Scholar]
  55. Yuspa S. H., Ben T., Hennings H. The induction of epidermal transglutaminase and terminal differentiation by tumor promoters in cultured epidermal cells. Carcinogenesis. 1983 Nov;4(11):1413–1418. doi: 10.1093/carcin/4.11.1413. [DOI] [PubMed] [Google Scholar]
  56. 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]
  57. Zettergren J. G., Peterson L. L., Wuepper K. D. Keratolinin: the soluble substrate of epidermal transglutaminase from human and bovine tissue. Proc Natl Acad Sci U S A. 1984 Jan;81(1):238–242. doi: 10.1073/pnas.81.1.238. [DOI] [PMC free article] [PubMed] [Google Scholar]

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