PaCDase enhances TNF-α gene expression via S1P and S1P receptors in 3D keratinocytes.

<p>(<i>A</i>) PaCDase induces TNF-α gene expression in 3D keratinocytes. Nitrocellulose filters with 1 mU/ml (60 ng/ml) PaCDase, 60 ng/ml H97A/H99A-PaCDase (H97A/H99A), or 60 ng/ml heat-inactivated PaCDase (heated), with or without 0.1% Triton X-100, were placed onto the stratum corneum. The cells were incubated for 24 h, and TNF-α mRNA was assayed by quantitative real-time RT-PCR. Each bar represents the mean±SD of 10 independent experiments. **P<0.01. (<i>B</i>) Sphingosine and S1P enhance TNF-α gene expression. Nitrocellulose filters with 1 mU/ml PaCDase, 5 µM sphingosine, phytosphingosine, C14 sphingoid base, S1P, α-hydroxy myristic acid, or 0.1% DMSO (solvent control) in Tris-buffered saline containing 0.1% Triton X-100 were placed onto the stratum corneum. The cells were incubated for 24 h, and TNF-α mRNA was assayed by quantitative real-time RT-PCR and normalized relative to a RNA encoding an S18 ribosomal protein gene. Each bar represents the mean±SD of 5 independent experiments. **P<0.01; ***P<0.001. (<i>C</i>) Involvement of SphK and S1P receptor. Nitrocellulose filters with 1 mU/ml PaCDase in the absence or presence of 1 µM VPC 23019, 10 µM SphK inhibitor, or 40 µM curcumin in Tris-buffered saline containing 0.1% Triton X-100 were placed onto the stratum corneum. The cells were incubated for 24 h, and TNF-α mRNA was assayed by quantitative real-time RT-PCR. Each bar represents the mean±SD of 5 independent experiments. *P<0.05; **P<0.01; ***P<0.001.