438 Activation of protease-activated receptor 2 leads to impairment of keratinocyte differentiation and tight junction integrity

Abstract

Protease-Activated Receptor 2 (PAR2) is a transmembrane G-protein-coupled receptor that can be activated by endogenous serine proteases and by environmental proteases (e.g. bacteria products, dust mites, allergens). Increased proteases activity has been shown in atopic skin. Activation of PAR2 has been associated to inflammation, altered epidermal barrier function (e.g. increased proteolysis of filaggrin) and pruritus. PAR2 activation has been observed to impair epithelial tight junctions (TJ) function; however, this had not yet been investigated in primary human keratinocytes (PHK). We hypothesized that activation of PAR2 plays a role in epidermal barrier impairment in atopic skin. Goal of this study was to investigate in vitro the effect of PAR2 activation on TJ function and composition. Additionally, we investigated the effect of PAR2 on PHK terminal differentiation as this is critical for proper barrier formation. PHK were differentiated in high-Ca+2 media in the presence of the selective PAR2 agonist SLIGKV-NH2 or reverse peptide as control (100 μM). TJ integrity was assessed by trans-epithelial electrical resistance (TEER). Expression of epidermal barrier components was evaluated at the RNA (qPCR) and protein (e.g. immunostaining) level. We first confirmed PAR2 expression in our culture model and found it increased with PHK differentiation. Notably, SLIGKV-NH2 reduced TEER peak (p < 0.01; n=5; 96hours) and this was associated with reduced expression of occludin and ZO-1 (qPCR, p < 0.05; n=4; 72hours). Consistently, prominent punctate and disrupted occludin staining pattern was observed in SLIGKV-NH2 treated PHK, in contrast to the sharp linear staining observed in the control. SLIGKV-NH2 also reduced expression of filaggrin and loricrin (qPCR, p < 0.01; n=4; 72hours); but no significant effect was observed for claudin-1 or e-cadherin expression. Our studies revealed that PAR2 might contribute to epidermal barrier impairment of atopic skin, by compromising both TJ and stratum corneum integrity. Strategy to block PAR2 downstream signaling could result in much needed therapeutic interventions for atopic dermatitis by targeting both barrier and inflammation.