Platelet activating factor stimulates arachidonic acid release in differentiated keratinocytes via arachidonyl non-selective phospholipase A2

Abstract

Platelet activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is known to be present in excess in psoriatic skin, but its exact role is uncertain. In the present study we demonstrate for the first time the role of group VI PLA(2) in PAF-induced arachidonic acid release in highly differentiated human keratinocytes. The group IValpha PLA(2) also participates in the release, while secretory PLA(2)s play a minor role. Two anti-inflammatory synthetic fatty acids, tetradecylthioacetic acid and tetradecylselenoacetic acid, are shown to interfere with signalling events upstream of group IValpha PLA(2) activation. In summary, our major novel finding is the involvement of the arachidonyl non-selective group VI PLA(2) in PAF-induced inflammatory responses.

Fig. 1

PAF induces AA and OA release in differentiated keratinocytes. a Initial cell state. Fold induction of differentiation specific marker genes in proliferating vs post-confluent HaCaT cells. (N = 2, result shown from one representative experiment). b Dose–response relationship for PAF stimulation of HaCaT cells. The response is measured as fold induction of [³H] arachidonic acid and [¹⁴C] oleic acids compared to the unstimulated control. PAF exposure time was 60 min. (All dose–response data in cell culture have been statistically validated using one way ANOVA at the 95% confidence level, and the results shown are representative of at least three consecutive experiments, using at least three parallel samples in each experiment)

Fig. 2

Different PLA₂ inhibitors reduce PAF-induced AA and OA release. a In vitro cPLA₂ activity assay on lysate from HaCaT cells pre-treated with 25 μM MAFP (90 min) and stimulated by 20 μM PAF (60 min) prior to lysis. [95% significance indicated by asterisk (compared to unstimulated control) and Δ (compared to PAF-stimulated sample) as determined by Student’s t test]. Results shown are representative of at least three consecutive experiments, using at least three parallel samples in each experiment. Release assay of [³H] arachidonic acid and [¹⁴C] oleic acids compared to the unstimulated control (in %). Stimulus is 20 μM PAF for 60 min. b Dose–response inhibition by MAFP (pretreatment time 90 min.), c inhibition by indoxam (pretreatment time 90 min), d dose–response inhibition by PACOCF₃ (pretreatment time 15 min). Statistical testing and number of experiments as for Fig. 1b

Fig. 3

Tetradecylthioacetic acid and tetradecylselenoacetic acid inhibit arachidonic acid release. The response is measured as % release of [³H] arachidonic acid and [¹⁴C] oleic acids compared to the unstimulated control. Pretreatment time for all inhibitors is 90 min. Effects on the PAF-induced response (left) (20 μM PAF, 60 min). Dose–response inhibition by a TTA and b TSA. c Dose–response result for Palmitic acid. PA acts as a control, since TTA and TSA are PA derivatives. Effects on the calcium ionophore (A23187)-induced response (right) (1 μM A23187, 60 min). d Dose–response inhibition by MAFP, e dose–response inhibition by TSA. Statistical testing and number of experiments as for Fig. 1b

Fig. 4

Tetradecylthioacetic acid and tetradecylselenoacetic acid point of action in AA-release is upstream of group IVα PLA₂. a In vitro activity assay applying inhibitors directly to lysate from group IVα PLA₂ overexpressing insect cells (95% significance compared to control indicated by asterisk as determined by Student’s t test). b Simultaneous application of TTA and PACOCF₃ is additive. The response is measured as % release of [³H] arachidonic acid compared to the unstimulated control. Stimulus is 20 μM PAF for 60 min. Pretreatment time for TTA is 90 min, PACOCF₃ was added 15 min, before PAF stimulation. Results shown are representative of at least three consecutive experiments, using at least three parallel samples in each experiment