Study On The Mechanism Of Trehalose-induced Autophagy In Human Keratinocytes And The Effect Of Trehalose Against Ultraviolet Damage

Ultraviolet is one common stressor of skin.Autophagy is a self-digestive pathway to maintain cellular homeostasis.It is unclear how autophagy is regulated by ultraviolet exposure in epidermal keratinocytes.Here,we found that UVB radiation inhibits basal autophagy flux in human keratinocytes.Moreover,mechanistic target of rapamycin(MTOR)signaling is contradictorily decreased,suggesting the MTOR-independentresponse in keratinocytes.This study provided a linkage of autophagy and skin disorders associated with ultraviolet.The mechanistic target of rapamycin(MTOR)protein is a crucial signaling regulator in mammalian cells that is extensively involved in cellular biology.The function of MTOR signaling in keratinocytes remains unclear.In this study,we detected the MTOR signaling and autophagy response in the human keratinocyte cell line HaCaT and human epidermal keratinocytes(HEKs)treated with MTOR inhibitors.Moreover,we detected the impact of MTOR inhibitors on keratinocytes exposed to the common carcinogenic stressors ultraviolet B(UVB)and UVA radiation.As a result,keratinocytes were sensitive to the MTOR inhibitors rapamycin,everolimus,Torin 1 and pp242,but the regulation of MTOR downstream signaling was distinct.Next,autophagy induction only was observed in HaCaT cells treated with rapamycin.Furthermore,we found that MTOR signaling was insensitive to UVB but sensitive to UVA radiation.UVB treatment also had no impact on the inhibition of MTOR signaling by MTOR inhibitors.Finally,MTOR inhibition by rapamycin,everolimus or pp242 did not affect the series of biological events in keratinocytes exposed to UVB,including the down-regulation of BIP and PERK,activation of Histone H2A and JNK and cleavage of caspase 3 and PARP.Our study demonstrated that MTOR inhibition in keratinocytes cannot always induce autophagy,and the MTOR pathway does not play a central role in the UVB-triggered cellular response.Trehalose is a natural disaccharide that is found in a diverse range of organisms but not in mammals.Autophagy is a process in which double-membrane autophagosomes are formed;this process mediates the sequestration,lysosomal delivery and degradation of proteins and organelles.Studies have shown that trehalose exerts beneficial effects through inducing autophagy in mammalian cells.However,whether trehalose or other saccharides can activate autophagy in keratinocytes is unknown.Here,we found that trehalose treatment increased the LC3-Ⅰ to LC3-Ⅱ conversion,acridine orange-stained vacuoles and GFP-LC3B(LC3B protein tagged with green fluorescent protein)puncta in the HaCaT human keratinocyte cell line,indicating autophagy induction.MTOR-independent autophagy induction was also observed in HaCaT cells treated with sucrose or raffinose but not in glucose,maltose or sorbitol treated HaCaT cells,indicating that autophagy induction was not a general property of saccharides.Finally,although trehalose treatment had an inhibitory effect on cell proliferation,it had the cytoprotective effect and inhibition on abnormal cell proliferation in cells exposed to UVB radiation.Our study provides new insight into the saccharide-mediated regulation of autophagy in keratinocytes.