ABSTRACT
House dust mites (HDMs) are known to trigger chronic inflammation through Toll-like receptors (TLRs) and their signalling cascades. In this study, we found that TLR2 ligation by HDMs induced the activation of dual oxidase 2 (Duox2) and nuclear factor-κB (NF-κB), leading to the production of pro-inflammatory cytokines in human keratinocytes. Stimulation of human keratinocytes with HDMs resulted in increases in interleukin-8 (IL-8) and chemokine (C-C motif) ligand 20 (CCL20) levels. However, pro-inflammatory cytokine production was abolished in keratinocytes transfected with TLR2 siRNA, indicating that HDM-induced cytokine production was mediated via TLR2 signalling. We also examined the function of Duox1/2 isozymes, which are primarily expressed in keratinocytes, in HDM-mediated pro-inflammatory cytokine production. Human keratinocytes transfected with control siRNA or Duox1 siRNA showed no inhibition of IL-8 or CCL20 production in response to HDMs, whereas the silencing of Duox2 expression resulted in a failure to induce cytokine production. Moreover, the phosphorylation and nuclear localization of RelA/p65, a component of NF-κB, were induced by HDMs in human keratinocytes. Transfection of human keratinocytes with TLR2 siRNA or Duox2 siRNA resulted in the complete abolishment of RelA/p65 nuclear localization in response to HDMs. Taken together, these results indicate that the HDM-dependent TLR2-Duox2 signalling axis indeed promotes NF-κB activation, which induces IL-8 and CCL20 production and mediates epidermal keratinocyte inflammation.
Subject(s)
Cytokines/biosynthesis , Dermatophagoides pteronyssinus/immunology , Dermatophagoides pteronyssinus/pathogenicity , Inflammation Mediators/metabolism , Keratinocytes/immunology , Keratinocytes/metabolism , NADPH Oxidases/metabolism , Animals , Cells, Cultured , Chemokine CCL20/biosynthesis , Dual Oxidases , Enzyme Activation , Gene Knockdown Techniques , Humans , Interleukin-8/biosynthesis , NADPH Oxidases/antagonists & inhibitors , NADPH Oxidases/genetics , Reactive Oxygen Species/metabolism , Signal Transduction , Toll-Like Receptor 2 , Transcription Factor RelA/metabolismABSTRACT
Testosterone is an endocrine hormone with functions in reproductive organs, anabolic events, and skin homeostasis. We report here that GPRC6A serves as a sensor and mediator of the rapid action of testosterone in epidermal keratinocytes. The silencing of GPRC6A inhibited testosterone-induced intracellular calcium ([Ca(2+)]i) mobilization and H2O2 generation. These results indicated that a testosterone-GPRC6A complex is required for activation of Gq protein, IP3 generation, and [Ca(2+)]i mobilization, leading to Duox1 activation. H2O2 generation by testosterone stimulated the apoptosis of keratinocytes through the activation of caspase-3. The application of testosterone into three-dimensional skin equivalents increased the apoptosis of keratinocytes between the granular and stratified corneum layers. These results support an understanding of the molecular mechanism of testosterone-dependent apoptosis in which testosterone stimulates H2O2 generation through the activation of Duox1.
