Phospholipase d signaling and extracellular signal-regulated kinase-1 and -2 phosphorylation (activation) are required for maximal phorbol ester-induced transglutaminase activity, a marker of keratinocyte differentiation.
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.
Bollag
Wendy B
WB
Institute of Molecular Medicine and Genetics/CB-2803, Medical College of Georgia, 1120 15th St., Augusta, GA 30912-2630, USA. wbollag@mail.mcg.edu
Zhong
Xiaofeng
X
Dodd
M Ernest
ME
Hardy
David M
DM
Zheng
Xiangjian
X
Allred
W Thomas
WT
D000818:Animals; D015415:Biomarkers; D002454:Cell Differentiation; D002478:Cells, Cultured; D004789:Enzyme Activation; D015603:Keratinocytes / Q000166:cytology*; D051379:Mice; D008813:Mice, Inbred ICR; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D010739:Phospholipase D / Q000037:antagonists & inhibitors / Q000502:physiology*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000502:physiology; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D011503:Transglutaminases / Q000378:metabolism*
D015415:Biomarkers; D011503:Transglutaminases; D011493:Protein Kinase C; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D010739:Phospholipase D; D013755:Tetradecanoylphorbol Acetate