Abstract
The hydroxylated benzene metabolite hydroquinone (HQ) is mainly generated from benzene, an important industrial chemical, and is also a common dietary component. Although numerous reports have addressed the tumorigenesis-inducing effects of HQ, few papers have explored its molecular regulatory mechanism in immunological responses. In this study we characterized Akt (protein kinase B)-targeted regulation by HQ and its derivatives, in suppressing inflammatory responses using cellular, molecular, biochemical, and immunopharmacological approaches. HQ down-regulated inflammatory responses such as NO production, surface levels of pattern recognition receptors, and cytokine gene expression with IC(50) values that ranged from 5 to 10 microm. HQ inhibition was mediated by blocking NF-kappaB activation via suppression of its translocation pathway, which is composed of Akt, I kappaB alpha kinase beta, and I kappaB alpha. Of the targets in this pathway, HQ directly targeted and bound to the sulfhydryl group of Cys-310 of Akt and sequentially interrupted the phosphorylation of both Thr-308 and Ser-473 by mediation of beta-mercaptoethanol, according to the liquid chromatography/mass spectroscopy analysis of the interaction of HQ with an Akt-derived peptide. Therefore, our data suggest that Akt and its target site Cys-310 can be considered as a prime molecular target of HQ-mediated immunosuppression and for novel anti-Akt-targeted immunosuppressive drugs.
Highlights
Inflammation is one of the innate immunity responses and a multiple step process that is mediated by activated inflammatory or immune cells
(NO) and prostaglandin E2), which are generated by activated inducible nitric oxide synthase and cyclooxygenase-2, and various chemokines and cytokines, such as tumor necrosis factor-␣ (TNF-␣),3 interleukin (IL)-1, IL-6, and IL-12 [1, 2]
Phospho- or total antibodies to p85, 3-phosphoinositide-dependent kinase 1 (PDK1), Akt (Thr-308 and Ser-473), extracellular signal-regulated kinase (ERK), ERK kinase (MEK), p38, c-Jun N-terminal kinase (JNK), IB␣ kinase (IKK), IB␣, p65, myelin basic protein (MBP), ␥-tubulin, and -actin were purchased from Cell Signaling (Beverly, MA), Santa Cruz Biotechnology (Santa Cruz, CA), or Upstate Biotechnology, Inc. (Lake Placid, NY)
Summary
Hydroquinone, tert-butyl hydroquinone, resorcinol, curcumin, indomethacin, arachidonic acid (AA), 1,4-dithiothreitol (DTT), L-cysteine, N-acetyl-L-cysteine, ␣-tocopherol, phorbol 12-myristate 13-acetate, D-galactosamine, prednisolone, and LPS (Escherichia coli 0111:B4) were purchased from Sigma. Fetal bovine serum and RPMI1640 were obtained from Invitrogen. Hae Young (Pusan National University, Pusan, Korea). Crosstide [14] and Suntide [15] were synthesized from Peptron (Daejeon, Korea). Phospho- or total antibodies to p85, 3-phosphoinositide-dependent kinase 1 (PDK1), Akt (protein kinase B) (Thr-308 and Ser-473), extracellular signal-regulated kinase (ERK), ERK kinase (MEK), p38, c-Jun N-terminal kinase (JNK), IB␣ kinase (IKK), IB␣, p65, myelin basic protein (MBP), ␥-tubulin, and -actin were purchased from Cell Signaling (Beverly, MA), Santa Cruz Biotechnology (Santa Cruz, CA), or Upstate Biotechnology, Inc. Alexa 488-conjugated secondary antibody was obtained from Invitrogen. ICR and C57BL/6 male mice (6 – 8 weeks old, 17–21 g) were obtained from Daehan Biolink (Chungbuk, Korea) and maintained in plastic cages under conventional conditions.
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