255 Keratinocytes isolated from dark or light pigmented skin showed different degrees of tight junction impairment after PAR2 activation in-vitro

Abstract

Protease activated receptor 2 (PAR2) is a transmembrane receptor with a tethered extracellular amino-terminus small peptide which acts as an activating ligand after cleavage. In the skin, PAR2 has extensively documented effects in promoting Th2-inflammation, skin barrier impairment and pruritus. Increased PAR2 expression was found in epidermal nerve fibers and keratinocytes in Atopic Dermatitis (AD) skin. In this study we aim to investigate the effect of PAR2 on Tight Junction (TJ) function and composition in primary human keratinocytes (PHK) isolated from dark or light pigmented foreskin. PHK were differentiated in high-Ca+2 media in the presence of the selective PAR2 agonist (SLIGKV-NH2) or reverse peptide as control. TJ integrity was assessed by trans-epithelial electrical resistance (TEER) and permeability to Na-Fluorescein. Expression of PAR2 and TJ components was evaluated at the RNA level. We confirmed greater expression of PAR2 in dark vs light-PHK. Both dark and light PHK differentiating in the context of PAR2 activation (100 μM) had a reduced TJ function resulting in reduced TEER and increased permeability to Na-Fluorescein (p≤0.05, n=5/each group). However, the degree of perturbation was greater for light-PHK as it compared to dark-PHK. Reduction in TEER after PAR2 activation was 40.8% in light-PHK vs 16.6% in dark-PHK (AUC 72-144 hours, p=0.01, n=5/each group). Also, a dose response effect was observed only in light-PHK, with significant TEER reduction after 50 μM PAR2 (p=0.03 at 120 hours). Notably, we observed a greater effect of PAR2 activation on the downregulation of CLDN1 and occludin mRNA in light-PHK. Our data highlights differences between keratinocytes of different ethnic backgrounds in TJ regulation through PAR2 activation. As we transition toward a better understating of AD phenotype, it is mandatory to keep in consideration and investigate intrinsic ethnic difference in skin barrier regulation that could be relevant to AD pathogenesis/treatment.