ER stress induced by ER calcium depletion and UVB irradiation regulates tight junction barrier integrity in human keratinocytes

Abstract

BackgroundEndoplasmic reticulum (ER) calcium depletion-induced ER stress is a crucial signal for keratinocyte differentiation and barrier homeostasis, but its effects on the epidermal tight junction (TJ) have not been characterized. Ultraviolet B (UVB) causes ER calcium release in keratinocytes and disrupts epidermal TJ, however, the involvement of ER stress in the UVB-induced TJ alterations remains unknown. ObjectivesTo investigate the effect of ER stress by pharmacological ER calcium depletion or UVB on the TJ integrity in normal human epidermal keratinocytes (NHEK). MethodsNHEK were exposed to ER calcium pump inhibitor thapsigargin (Tg) or UVB. ER stress markers and TJ molecules expression, TJ and F-actin structures, and TJ barrier function were analyzed. ResultsTg or UVB exposure dose-dependently triggered unfolded protein response (UPR) in NHEK. Low dose Tg induced the IRE1α-XBP1 pathway and strengthened TJ barrier. Contrary, high dose Tg activated PERK phosphorylation and disrupted TJ by F-actin disorganization. UVB disrupted TJ and F-actin structures dose dependently. IRE1α RNase inhibition induced or exacerbated TJ and F-actin disruption in the presence of low dose Tg or UVB. High dose Tg increased RhoA activity. 4-PBA or Rho kinase (ROCK) inhibitor partially prevented the disruption of TJ and F-actin following high dose Tg or UVB. ConclusionsER stress has bimodal effects on the epidermal TJ depending on its intensity. The IRE1α pathway is critical for the maintenance of TJ integrity during mild ER stress. Severe ER stress-induced UPR or ROCK signalling mediates the disruption of TJ through cytoskeletal disorganization during severe ER stress.