Carcinogenesis, Vol. 20, No. 4, 569-576,
April 1999
© 1999 Oxford University Press
Sustained phospholipase D activation is associated with keratinocyte differentiation
Program in Cell Signaling, Institute of Molecular Medicine and Genetics, Medical College of Georgia, 1120 15th Street, Augusta, GA 30912-2630, USA
Our previous results and data in the literature have suggested a potential role for phospholipase D (PLD) in the regulation of epidermal keratinocyte growth and differentiation. Therefore, we investigated the effect of agents reported to modulate keratinocyte growth and differentiation on PLD activation. The purported protein kinase C (PKC) `inhibitor', staurosporine (Stsp), has been reported to activate PKC in keratinocytes, eliciting many of the same effects as active tumor promoters such as 12-O-tetradecanoylphorbol-13-acetate (TPA). Stsp also induces a programmed pattern of differentiation similar to that seen in keratinocytes in vivo; TPA, on the other hand, appears to preferentially elicit markers consistent with late (granular) differentiation. In contrast, bradykinin is reported to stimulate keratinocyte proliferation. We found that these three agents had different effects on PLD activation in primary mouse epidermal keratinocytes. TPA increased PLD activity acutely and in a sustained fashion. In contrast, Stsp did not acutely activate PLD and inhibited acute TPA-induced activation of PLD. However, treatment of keratinocytes with Stsp for longer time periods (3–5 h) induced sustained PLD activation and this long-term effect was additive with that of TPA. Bradykinin activated PLD acutely but transiently. Both TPA and Stsp increased transglutaminase activity, a marker of late differentiation, whereas bradykinin had little or no effect on either cell proliferation or transglutaminase activity. These results suggest that a sustained activation of PLD is associated with the induction of keratinocyte differentiation. We hypothesize that PLD activity mediates late keratinocyte differentiation through generation of diacylglycerol and activation of specific PKC isoforms. Furthermore, we propose that the profound and immediate TPA-induced stimulation of PLD activity `drives' the keratinocytes to late differentiation steps. However, the less efficacious (and more gradual) sustained activation of PLD by Stsp may allow a patterned differentiation more like that observed in skin.
Abbreviations: BSA, bovine serum albumin; DAG, sn-1,2-diacylglycerol; FBS, fetal bovine serum; IC50, half-maximal inhibitory concentration; ITS, insulin transferrin and selenious acid; ITS+, ITS + linoleic acid/BSA; MEM, minimum essential medium; PA, phosphatidic acid; PEt, phosphatidylethanol; PLD, phospholipase D; PKC, protein kinase C; SDS, sodium dodecyl sulfate; SFKM, serum-free keratinocyte medium; Stsp, staurosporine; TCA, trichloroacetic acid; TPA, 12-O-tetradecanoylphorbol-13-acetate.
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