Molecular signaling of the HMGB1/RAGE axis contributes to cholesteatoma pathogenesis

Abstract

Cholesteatoma represents progressive expansion of the keratinizing squamous epithelium in the middle ear with subsequent chronic inflammation in subepithelial connective tissues. The hypothesis was tested that receptor for advanced glycation endproduct (RAGE) and its ligand, high-mobility box 1 (HMGB1), are overexpressed in cholesteatoma, and the RAGE/HMGB1 axis might contribute to its pathogenesis. Cholesteatoma samples (n = 36) and 27 normal skin specimens were studied by immunohistochemistry (IHC) for HMGB1 and RAGE expression. Effects of HMGB1 signaling on proliferation, migration, cytokine production, and apoptosis of human immortalized keratinocytes (HaCaTs) and normal keratinocytes were studied by quantitative reverse transcription (qRT)-PCR, IHC, Western blots, and flow cytometry after cell co-incubation with HMGB1. While all studied tissues expressed HMGB1, its expression was higher in cholesteatoma than in normal skin (p < 0.0001). All cases of cholesteatoma also showed elevated RAGE expression levels, and only 7/27 (26 %) of normal skin specimens were weakly positive for RAGE. Proliferation and migration of HaCaT cells incubated with HMGB1 were up-regulated (p < 0.05). HMGB1 also prevented HaCaT cell apoptosis and induced activation of several molecular signaling pathways in keratinocytes. The data suggest that in cholesteatoma, HMGB1 released from stressed or necrotic epithelial cells and binding to RAGE overexpressed in keratinocytes initiates molecular signaling that culminates in pro-inflammatory cytokine release and chronic inflammation.Key messageHMGB1 signaling engages multiple activation pathways in RAGE-positive keratinocytes.HMGB1 protects RAGE-positive keratinocytes from drug-induced apoptosis.Keratinocyte proliferation is controlled via RAGE and HMGB1 molecular signaling.Molecular signaling of the HMGB1/RAGE axis contributes to cholesteatoma pathogenesis.Electronic supplementary materialThe online version of this article (doi:10.1007/s00109-014-1217-3) contains supplementary material, which is available to authorized users.

High-mobility group box protein 1 expression in inflammatory diseases of the middle ear

“…The paper by Szczepanski et al 8 strengthens our hypothesis: by histological analysis they demonstrated that HMGB1 protein and its major receptor RAGE are higher expressed in cholesteatoma samples than in normal skin. In addition, in an ex vivo study they showed that HMGB1 prevents human immortalized keratinocyte (HaCaTs) cell apoptosis: HMGB1 release from damaged and/or necrotic cells and its binding to RAGE receptor is the molecular signaling for cellular hyperplasia, proliferation, and cholesteatoma growth.…”

“…The middle ear is endowed with several mechanisms of defense against invading pathogens, pollutants, and allergens: the anatomic characteristics of the Eustachian tube (ET) in the first years of life, the mucociliary apparatus of its mucosa, and the secreted mucus and its content of soluble chemical factors such as surfactant proteins, lactoferrin, interferon, and defensins. 8 …”

Chronic inflammation of middle ear cholesteatoma promotes its recurrence via a paracrine mechanism

“…TLR4 signalling can also be induced by the DAMPs high-mobility group box 1 proteins (HMGB1), Tenascin, bronectin [all18], S100A8, S100A9 [both89] or the heat shock proteins HSP 60 and HSP 70 [both90]. Interestingly HMGB1 [91], Tenascin [92], bronectin [5], S100A8, S100A9 [both93] and also HSP60 and HSP70 [both94] are known to be more abundant in cholesteatoma tissue compared to healthy skin. Interestingly, the DAMPs HMGB1 and Tenascin are also suspected to contribute to cholesteatoma pathogenesis [91, and 95 respectively].…”

HMGB1 Promotes Intraoral Palatal Wound Healing through RAGE-Dependent Mechanisms

“…Collagen components are a major part of oral mucosa development [28]. The macroscopic findings of wound closure were confirmed by histological assessment.…”

“…Although the distribution of HMGB1 in relation to wound healing has been observed in human skin [26] and cholesteatoma [28], studies dealing with palatal wound healing and the target mutation of Hmgb1 gene in vivo have not been previously performed. In our study, we found that HMGB1 expression in unwounded and wounded site of Hmgb1 +/− mice is very low compared to WT mice.…”