Sustained phospholipase D activation is associated with keratinocyte differentiation

Abstract

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-Otetradecanoylphorbol-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.

Cell wounding activates phospholipase D in primary mouse keratinocytes

“…We determined the ability of FIPI, an agent that inhibits both PLD1 and PLD2 ( 26 ), to inhibit PLD activity in intact keratinocytes by measuring the effect of FIPI pretreatment on PLD activation in response to the phorbol ester, 12-O -tetradecanoylphorbol 13-acetate (TPA). As shown previously ( 27 ), TPA stimulated PLD activity in keratinocytes, as measured by an approximate 4.5-fold increase in radiolabeled PEt levels ( Fig. 7 ).…”

Putative Conventional Protein Kinase C Inhibitor Gödecke 6976 [12-(2-Cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole] Stimulates Transglutaminase Activity in Primary Mouse Epidermal Keratinocytes

“…Subsequently, [ 3 H]thymidine (1 Ci/ml final concentration) was added, and the cells were incubated an additional 60 min. The radioactivity in 5% trichloroacetic acid-precipitable DNA was then quantified by liquid scintillation spectroscopy, following washing and solubilization in 0.3 M NaOH as in Jung et al (1999).…”

“…Keratinocytes were incubated for 24 h in SFKM containing the appropriate agents. After the cells were scraped into homogenization buffer [0.1 M Tris-acetate, pH 7.8, 2 g/ml aprotinin, 2 M leupeptin, 1 M pepstatin A, 0.2 mM EDTA, and 20 M 4-(2-aminoethyl)benzenesulfonyl fluoride] and subjected to one freeze-thaw cycle, transglutaminase activity was determined by monitoring the incorporation of [ 3 H]putrescine into casein as described in Jung et al (1999).…”

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

“…Inhibitor concentrations were selected based on previous data from our laboratory Shapiro et al, 2002), and all samples contained the same concentration of vehicle (dimethyl sulfoxide; DMSO). Phospholipids were extracted with chloroform/methanol and separated by thin layer chromatography as described in Jung et al (1999). Radiolabeled phosphatidylethanol, a unique marker of PLD activity (Thompson et al, 1991), was quantified by liquid scintillation spectrometry, also as described in Jung et al (1999).…”

“…Phospholipids were extracted with chloroform/methanol and separated by thin layer chromatography as described in Jung et al (1999). Radiolabeled phosphatidylethanol, a unique marker of PLD activity (Thompson et al, 1991), was quantified by liquid scintillation spectrometry, also as described in Jung et al (1999).…”