Effects of the selective protein kinase C inhibitor, Ro 31-7549, on the proliferation of cultured mouse epidermal keratinocytes

Wendy B. Bollag, Janet Ducote, Charles S. Harmon

Research output: Contribution to journalArticle

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Abstract

We have investigated the effects of Ro 31-7549, a selective protein kinase C (PKC) inhibitor, on DNA synthesis and proliferation in two primary mouse epidermal keratinocyte culture systems. In differentiating keratinocytes incubated in medium containing 10% serum and high calcium (approximately 0.5 mM), Ro 31-7549 blocked the inhibitory effect of the phorbol ester 12-0-tetradecanoyl-13-acetate (TPA) (a PKC activator) on keratinocyte DNA synthesis at 24 h [50% maximal response concentration (EC50) = 1μm], consistent with inhibition of PKC-mediated differentiation. Continuous treatment of the differentiative culture system with the PKC inhibitor resulted in a marked (fourfold) stimulation of [3H]thymidine incorporation at day 7 of exposure, with an EC50 of 0.25 μM. The potencies of these effects of Ro 31-7549 are comparable to that reported for inhibition of TPA-induced platelet 47-kD protein phosphorylation [50% inhibitory concentration (IC50)= 4.4 μm]. The time course of [3h]thymidine incorporation indicated that Ro 31-7549 did not directly stimulate DNA synthesis but instead prevented the loss of proliferative capacity associated with continued culture in this medium. Maximal stimulation (2.6 times) of DNA synthesis was observed on day 4, whereas DNA synthesis at day 1 was unaffected. In a highly proliferative culture system using serum-free medium containing 25μM calcium, TPA dose-dependently inhibited proliferation with an IC50 of approximately 0.3 nM. This antiproliferative effect of TPA was largely reversed by 0.1 μM Ro 31-7549. In the proliferative culture system, 0.75μM Ro 31-7549 also essentially reversed the inhibition of proliferation caused by switching to high (1.0 mM) calcium. These results suggest that the loss of proliferative capacity in differentiating epidermal keratinocyte cultures may be mediated, at least in part, by PKC.

Original languageEnglish (US)
Pages (from-to)240-246
Number of pages7
JournalJournal of Investigative Dermatology
Volume100
Issue number3
DOIs
StatePublished - Mar 1993

Fingerprint

Protein C Inhibitor
Protein Kinase Inhibitors
Keratinocytes
Protein Kinase C
DNA
Inhibitory Concentration 50
Calcium
Thymidine
Phosphorylation
Serum-Free Culture Media
Phorbol Esters
Platelets
Culture Media
Ro 31-7549
Acetates
Blood Platelets
Serum
Proteins

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Dermatology
  • Cell Biology

Cite this

Effects of the selective protein kinase C inhibitor, Ro 31-7549, on the proliferation of cultured mouse epidermal keratinocytes. / Bollag, Wendy B.; Ducote, Janet; Harmon, Charles S.

In: Journal of Investigative Dermatology, Vol. 100, No. 3, 03.1993, p. 240-246.

Research output: Contribution to journalArticle

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abstract = "We have investigated the effects of Ro 31-7549, a selective protein kinase C (PKC) inhibitor, on DNA synthesis and proliferation in two primary mouse epidermal keratinocyte culture systems. In differentiating keratinocytes incubated in medium containing 10{\%} serum and high calcium (approximately 0.5 mM), Ro 31-7549 blocked the inhibitory effect of the phorbol ester 12-0-tetradecanoyl-13-acetate (TPA) (a PKC activator) on keratinocyte DNA synthesis at 24 h [50{\%} maximal response concentration (EC50) = 1μm], consistent with inhibition of PKC-mediated differentiation. Continuous treatment of the differentiative culture system with the PKC inhibitor resulted in a marked (fourfold) stimulation of [3H]thymidine incorporation at day 7 of exposure, with an EC50 of 0.25 μM. The potencies of these effects of Ro 31-7549 are comparable to that reported for inhibition of TPA-induced platelet 47-kD protein phosphorylation [50{\%} inhibitory concentration (IC50)= 4.4 μm]. The time course of [3h]thymidine incorporation indicated that Ro 31-7549 did not directly stimulate DNA synthesis but instead prevented the loss of proliferative capacity associated with continued culture in this medium. Maximal stimulation (2.6 times) of DNA synthesis was observed on day 4, whereas DNA synthesis at day 1 was unaffected. In a highly proliferative culture system using serum-free medium containing 25μM calcium, TPA dose-dependently inhibited proliferation with an IC50 of approximately 0.3 nM. This antiproliferative effect of TPA was largely reversed by 0.1 μM Ro 31-7549. In the proliferative culture system, 0.75μM Ro 31-7549 also essentially reversed the inhibition of proliferation caused by switching to high (1.0 mM) calcium. These results suggest that the loss of proliferative capacity in differentiating epidermal keratinocyte cultures may be mediated, at least in part, by PKC.",
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