Abstract
Protein kinase C (PKC)-activating 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulates phospholipase D (PLD) activity in primary mouse epidermal keratinocytes. PLD catalyzes the hydrolysis of phosphatidylcholine to yield phosphatidic acid (PA), which can be dephosphorylated to produce PKC-activating diacylglycerol. In the presence of small amounts of a primary alcohol, PLD can instead produce novel phosphatidylalcohols at the expense of PA and diacylglycerol. Here, we have demonstrated that inhibiting PLD signal generation with 1-butanol reduced TPA-stimulated transglutaminase activity, a marker of keratinocyte differentiation. On the other hand, the structurally related tertiary alcohol tert-butanol, which cannot be used by PLD, had no effect on TPA-induced transglutaminase activity. Since TPA activates all conventional and novel PKC isoforms directly, yet cannot overcome 1-butanol-mediated inhibition, this result suggests that PLD mediates its effects on transglutaminase activity (and keratinocyte differentiation) through an effector enzyme system distinct from the conventional or novel PKC isoenzymes. Data in the literature suggest that PA can recruit Raf-1 to the membrane, where it can be activated and initiate the mitogen-activated protein kinase cascade that culminates in activation of extracellular signal-regulated kinase (ERK)-1 and -2. Indeed, we found that inhibition of ERK-1/2 phosphorylation (activation) inhibited TPA-induced transglutaminase activity. However, inhibition of PLD-mediated signal generation had only a small effect on TPA-elicited ERK-1/2 phosphorylation (activation), whereas inhibition of ERK-1/2 did not affect PLD activation, suggesting that these two pathways likely operate largely in parallel. Thus, our results suggest the independent involvement of the PLD and ERK-1/2 pathways in mediating transglutaminase activity and keratinocyte differentiation.
Footnotes
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↵1 Current address: Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN.
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This study was supported by the National Institutes of Arthritis, Musculoskeletal, and Skin Diseases Grant AR45212. This work was presented in part at the 64th Annual Meeting of the Society for Investigative Dermatology (Miami Beach, FL, May 2003) and the 37th Meeting of the Southeast Regional Lipid Conference (Cashiers, NC, November 2002).
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doi:10.1124/jpet.104.075622.
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ABBREVIATIONS: TPA, 12-O-tetradecanoyl phorbol 13-acetate; PKC, protein kinase C; PLD, phospholipase D; PA, phosphatidic acid; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; SFKM, serum-free keratinocyte medium; DMSO, dimethyl sulfoxide; BisI, bisindolylmaleimide I.
- The American Society for Pharmacology and Experimental Therapeutics
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