N-nitroso-N-methylurea and N-nitroso-N-ethylurea induce upregulation of cellular NF-κ B activity through protein kinase C-dependent pathway in human malignant keratinocytes

The upregulatory mechanism of cellular NF-κB activity by carcinogens, N -nitroso- N -methylurea (NMU) and N -nitroso- N -ethylurea (NEU) in human malignant keratinocytes was investigated. To elucidate the role of protein kinase C (PKC) in the upregulation of NF-κB by NMU and NEU, two known PKC inhibitors, staurosporine and H-7 were studied. Treatment of cells with PKC inhibitors decreased NF-κB activity in a dose responsive manner at concentrations of 20∼200 nM. Staurosporine (160 nM) and H-7 (200 nM) downregulated the cellular NF-κB activation up to 20 and 60% compared to the NF-κB activity that was upregulated by NMU (5 μM) and NEU (5 μM), respectively. These results indicated that the PKC activity was responsible for the upregulation of NF-κB activity. The level of phosphorylation of I-κBα, the predominant form of the I-κB family represented by NMU and NEU, was quantified. The relative amount of I-κBα phosphorylation (serines-32 and -36) determined using the cellular activation of signaling ELISA assay method showed that NMU (5 μM) and NEU (5 μM) increased the amount of I-κBα phosphorylation up to 17 and 10% compared to the control, respectively. The results demonstrate the upregulatory effect of NMU and NEU on cellular NF-κB activity in human keratinocytes via the protein kinase C-mediated pathway.