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BACKGROUND AND PURPOSEPungent constituents of ginger (Zingiber officinale) have beneficial effects on inflammatory pain and arthritic swelling. However, the molecular basis for these pharmacological properties is only partially understood. Here, we investigated the molecular target of 1-dehydro-[10]-gingerdione (D10G), one of the pungent constituents of ginger, that mediates its suppression of NF-kB-regulated expression of inflammatory genes linked to toll-like receptor (TLR)-mediated innate immunity. EXPERIMENTAL APPROACHRAW 264.7 macrophages or primary macrophages-derived from bone marrows of C57BL/6 or C3H/HeJ mice were stimulated with the TLR4 agonist LPS in the presence of D10G. Catalytic activity of inhibitory kB (IkB) kinase b (IKKb) was determined by a kinase assay and immunoblot analysis, and the expression of inflammatory genes by RT-PCR analysis and a promoter-dependent reporter assay. KEY RESULTSD10G directly inhibited the catalytic activity of cell-free IKKb. Moreover, D10G irreversibly inhibited cytoplasmic IKKb-catalysed IkBa phosphorylation in macrophages activated by TLR agonists or TNF-a, and also IKKb vector-elicited NF-kB transcriptional activity in these cells. These effects of D10G were abolished by substitution of the Cys 179 with Ala in the activation loop of IKKb, indicating a direct interacting site of D10G. This mechanism was shown to mediate D10G-induced disruption of NF-kB activation in LPS-stimulated macrophages and the suppression of NF-kB-regulated gene expression of inducible NOS, COX-2 and IL-6. CONCLUSION AND IMPLICATIONSThis study demonstrates that IKKb is a molecular target of D10G involved in the suppression of NF-kB-regulated gene expression in LPS-activated macrophages; this suggests D10G has therapeutic potential in NF-kB-associated inflammation and autoimmune disorders. AbbreviationsAP-1, activating protein 1; D10G, 1-dehydro-[10]-gingerdione; IkB, inhibitory kB; IKKb, IkB kinase b; iNOS, inducible NOS; PTN, parthenolide; TNFSF11, receptor activator of NF-kB ligand; SEAP, secretory alkaline phosphatase; TLR, toll-like receptor

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BACKGROUND AND PURPOSEcAMP as a second messenger stimulates expression of microphthalmia-associated transcription factor (MITF) or the tyrosinase gene in UVB-induced skin pigmentation. Diphenylmethylene hydrazinecarbothioamide (QNT 3-80) inhibits α-melanocytestimulating hormone (α-MSH)-induced melanin production in B16 murine melanoma cells but its molecular basis remains to be defined. Here, we investigated the mechanism underlying the amelioration of skin hyperpigmentation by QNT 3-80. EXPERIMENTAL APPROACHWe used melanocyte cultures with raised levels of cAMP and UVB-irradiated dorsal skin of guinea pigs for pigmentation assays. Immunoprecipitation, kemptide phosphorylation, fluorescence analysis and docking simulation were applied to elucidate a molecular mechanism of QNT 3-80. KEY RESULTSQNT 3-80 inhibited melanin production in melanocyte cultures with elevated levels of cAMP, including those from human foreskin. This compound also ameliorated hyperpigmentation in vivo in UVB-irradiated dorsal skin of guinea pigs. As a mechanism, QNT 3-80 directly antagonized cAMP binding to the regulatory subunit of PKA, nullified the dissociation and activation of inactive PKA holoenzyme in melanocytes and fitted into the cAMP-binding site on the crystal structure of human PKA under the most energetically favourable simulation. QNT 3-80 consequently inhibited cAMP-or UVB-induced phosphorylation (activation) of cAMP-responsive element-binding protein in vitro and in vivo, thus down-regulating expression of genes for MITF or tyrosinase in the melanogenic process. CONCLUSIONS AND IMPLICATIONSOur data suggested that QNT 3-80 could contribute significantly to the treatment of skin disorders with hyperpigmented patches with the cAMP-binding site of PKA as its molecular target.