Abstract
Protein kinase C, the major cellular receptor for tumour-promoting phorbol esters, has been suggested as playing a key role in the regulation of proliferation and differentiation of epidermal cells. In the present study, we investigated the effects of various well-characterized inhibitors of protein kinase C on proliferation and differentiation of SV 40-transformed and normal human keratinocytes. The drugs were found to inhibit cell proliferation in a dose-dependent manner, displaying similar effects in both cell types and reflecting their potencies in inhibiting purified protein kinase C. In contrast, keratinocyte differentiation induced by treatment with a calcium ionophore or spontaneously, i.e. by exposure of cells grown in the presence of low calcium concentration (0.06 mM) to normal calcium concentration (1.6 mM), was not inhibited by the compounds tested. The potent protein kinase C inhibitor, staurosporine, was found even to enhance cell differentiation. Therefore, the present study provides evidence that the classical protein kinase C pathway plays a critical role in the regulation of keratinocyte proliferation rather than in calcium-induced differentiation.
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References
Blumberg PM (1988) Protein kinase C as the receptor for the phorbol ester tumor promotors. Cancer Res 48: 1–8
Borenfreund E, Puerner JA (1984) A simple quantitative procedure using monolayer cultures for cytotoxicity assays (HTD/NR-90). J Tissue Culture Methods 9: 7–9
Castagna M, Takai Y, Kaibuchi K, Sano K, Kikkawa U, Nishizuka Y (1982) Direct activation of calcium-activated, phospholipid-dependent protein kinase by tumor-promoting phorbol esters. J Biol Chem 257: 7847–7851
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
Dlugosz AA, Yuspa SH (1993) Coordinate changes in gene expression which mark the spinous to granular cell transition in epidermis are regulated by protein kinase C. J Cell Biol 120: 217–225
Dotto GP, Gilman MZ, Maruyama M, Weinberg RA (1986) c-myc and c-fos expression in differentiating mouse primary keratinocytes. EMBO J 5: 2853–2857
Dunn JA, Blumberg PM (1983) Specific binding of [20-3H]12-deoxyphorbol 13-isobutyrate to phorbol ester receptor subclasses in mouse skin particulate preparations. Cancer Res 43: 4632–4637
Fisher GJ, Harris VA, Voorhees JJ (1987) Partial purification and characterization of protein kinase C from adult human epidermis. J Invest Dermatol 89: 484–488
Fürstenberger G, Berry DL, Sorg B, Marks F (1981) Skin tumor promotion by phorbol esters is a two-stage process. Proc Natl Acad Sci USA 78: 7722–7726
Hawley-Nelson P, Stanley JR, Schmidt J, Gullino M, Yuspa SH (1982) The tumor promotor, 12-O-tetradecanoyl-phorbol-13-acetate accelerates keratinocyte differentiation and stimulates growth of an unidentified cell type in cultured human epidermis. Exp Cell Res 137: 155–167
Hegemann L, Fruchtmann R, Bonnekoh B, Schmidt BH, Traber J, Mahrle G, Müller-Peddinghaus R, van Rooijen LAA (1991) Effects of tiflucarbine as a dual protein kinase C/calmodulin antagonist on proliferation of human keratinocytes and release of reactive oxygen species from human leukocytes. Arch Dermatol Res 283: 456–460
Hegemann L, Mahrle G (1991) Biochemical pharmacology of protein kinase C and its relevance for dermatology. In: Mukhtar H (ed) Pharmacology of the skin. CRC Press, Boca Raton, pp 357–368
Hegemann L, Bonnekoh B, Rooijen LAA van, Mahrle G (1992a) Antiproliferative effects of protein kinase C inhibitors in human keratinocytes. J Dermatol Sci 4: 18–25
Hegemann L, Wevers A, Webster GF, Mahrle G, Uitto J (1992b) Protein kinase C subtypes in human skin (abstract). J Invest Dermatol 98: 584
Hennings H, Michael D, Cheng C, Steinert P, Holbrook KA, Yuspa SH (1980) Calcium regulation of growth and differentiation of mouse epidermal cells in culture. Cell 19: 245–254
Kikkawa U, Nishizuka Y (1986) The role of protein kinase C in transmembrane signalling. Annu Rev Cell Biol 2: 149–178
King I, Mella SL, Sartorelli AC (1986) A sensitive method to quantify the terminal differentiation of cultured epidermal cells. Exp Cell Res 167: 252–256
Melloni E, Pontremoli S, Viotti PL, Patrone M, Marks PA, Rifkind RA (1989) Differential expression of protein kinase C isozymes and erythroleukemia cell differentiation. J Biol Chem 264: 18414–18418
Moscat J, Fleming TP, Molloy CJ, Lopez-Barahona M, Aaronson SA (1989) The calcium signal for Balb/MK keratinocyte terminal differentiation induces sustained alterations in phosphoinositide metabolism without detectable protein kinase C activation. J Biol Chem 264: 11228–11235
Nishizuka Y (1984) The role of protein kinase C in cell surface signal transduction and tumour promotion. Nature 308: 693–697
Nishizuka Y (1989) The family of protein kinase C for signal transduction. J Am Med Assoc 262: 1826–1833
Ponec M, Kempenaar J, De Kloet ER (1981) Corticoids and cultured human epidermal keratinocytes: Specific intracellular binding and clinical efficacy. J Invest Dermatol 76: 211–214
Ponec M, Weerheim A, Kempenaar J, Boonstra J (1988) Proliferation and differentiation of human squamous carcinoma cell lines and normal keratinocytes: Effects of epidermal growth factor, retinoids, and hydrocortisone. In Vitro Cell Dev Biol 24: 764–770
Rheinwald JG, Green H (1975) Serial cultivation of strains of human epidermal keratinocytes: The formation of keratinizing colonies from single cell. Cell 6: 331–334
Rosenbach T, Liesegang C, Haase I, Binting S, Czarnetzki B (1993) Alterations of inositol phosphate formation during HaCaT keratinocyte proliferation and differentiation (abstract). J Invest Dermatol 100: 444
Rüegg UT, Burgess GM (1989) Staurosporine, K-252 and UCN-01: Potent but nonspecific inhibitors of protein kinases. Trends Pharmacol Sci 10: 218–220
Sako T, Tauber AI, Jeng AY, Yuspa SH, Blumberg PM (1988) Contrasting action of staurosporine, a protein kinase C inhibitor, on human neutrophils and primary mouse epidermal cells. Cancer Res 48: 4646–4650
Shoji M, Vogler WR, Kuo JF (1985) Inhibition of phospholipid /Ca2+-dependent protein kinase and phosphorylation of leukemic cell proteins by CP-46,665-1, a novel antineoplastic lipoidal amine. Biochem Biophys Res Commun 127: 590–595
Snoek GT, Boonstra J, Ponec M, De Laat SW (1987) Phorbol ester binding and protein kinase C activity in normal and transformed human keratinocytes. Exp Cell Res 172: 146–157
Sun TT, Green H (1976) Differentiation of the epidermal keratinocyte in cell culture: Formation of the cornified envelope. Cell 9: 511–521
Tamaoki T, Nomoto H, Takahashi I, Kato Y, Morimoto M, Tomita F (1986) Staurosporine, a potent inhibitor of phospholipid/Ca++ dependent protein kinase. Biochem Biophys Res Commun 135: 397–402
Taylor-Papadimitriou J, Purkis P, Lane EB, McKay IA, Chang SE (1982) Effects of SV 40-transformation on the cytoskeleton and behavioural properties of human keratinocytes. Cell Differ 11: 169–180
Wirth PJ, Yuspa SH, Thorgeirsson SS, Hennings H (1987) Induction of common patterns of polypeptide synthesis and phosphorylation by calcium and 12-O-tetradecanoyl-phorbol-13-acetate in mouse epidermal cell culture. Cancer Res 47: 2831–2838
Wooten MW (1992) Differential expression of PKC isoforms and PC12 cell differentiation. Exp Cell Res 199: 111–119
Yuspa SH, Ben T, Hennings H (1983) The induction of epidermal transglutaminase and terminal differentiation by tumor promotors in cultured epidermal cells. Carcinogenesis 4: 1413–1418
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Hegemann, L., Kempenaar, J. & Ponec, M. The involvement of protein kinase C in proliferation and differentiation of human keratinocytes — an investigation using inhibitors of protein kinase C. Arch Dermatol Res 286, 278–284 (1994). https://doi.org/10.1007/BF00387601
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DOI: https://doi.org/10.1007/BF00387601