The Effect of TFEB-Induced Autophagy on TGF-β1 Secretion of Keratinocyte

In present study, We investigated the effect of TFEB-induced autophagy on TGF-β1 secretion of keratinocytes and its possible mechanism. Human cutaneous KC was divided into control group, serum stimulation group, serum stimulation+TUDCA group, serum stimulation+TFEB siRNA group, serum stimulation+NC siRNA group and serum stimulation+chloroquine group. The secretion of TGF-β1 in keratinocytes was detected by ELISA kit. The expression of p-PERK, GRP78, TFEB, LC3, LAMP1, p-elF2α, CHOP and caspase-3 in keratinocytes was studied by Western blot. And the expression of α-SMA and COL I in firboblasts cultured with conditioned culture medium was also studied by Western blot. Inhibiting TFEB expression of keratinocytes by RNA interference, the secretion of TGF-β1 and the expression of p-elF2α, CHOP and caspase-3 was detected. And the expression of α-SMA and COL I in firboblasts was also studied by Western blot. After serum stimulation, co-localization of TGF-β1 and LAMP1, TGF-β1 and LC3 in keratinocytes was detected by immunofluorescence staining. After the addition of chloroquine, an autophagic lysosome pathway inhibitor, the co-localization of Rab8a and TGF-β1, Rab8a and LAMP1 in keratinocytes was detected by immunofluorescence staining. Compared with control group, serum stimulation can induce TGF-β1 secretion of keratinocytes, causing cell endoplasmic reticulum stress (increase GRP78, p-PERK expression, P<0.01), increase cell autophagy level (increase TFEB, LC3 II, LAMP1 expression, P<0.01) and increase the α- SMA, COL I protein expression of fibroblasts (P<0.01). After the addition of Sulfonic acid deoxycholic acid, an endoplasmic reticulum stress inhibitor, compared with serum stimulus group, the expression of GRP78 and p-PERK (P<0.05) decreased and the expression of TFEB, LC3 II and LAMP1 (P<0.05) in keratinocytes also decreased. After inhibiting TFEB expression of keratinocytes by RNA interference, compared with serum stimulus group, the secretion of TGF-β1 decreased obviously (P<0.01), and the expression of p-elF2α, CHOP and caspase-3 (P<0.01) in keratinocytes increased and the expression of α- SMA, COL I (P<0.01) in fibroblasts decreased significantly. Immunofluorescence showed that the co-localization level of TGF-β1 and LAMP1 (P<0.01), TGF-β1 and LC3 (P<0.01) in KC was significantly enhanced after serum stimulation. After addition of chloroquine, the co-localization level of Rab8a and TGF-β1 (P<0.05), Rab8a and LAMP1 (P<0.01) was significantly reduced, and the secretion of TGF-β1 was reduced (P<0.05). We concluded that TFEB-mediated autophagy reduces protein load in the endoplasmic reticulum and inhibits apoptosis-related caspase protein activation by clearing misfolded proteins and participating in TGF-β1 secretion, thereby reducing keratinocytes damage.

CSTR: 32200.14.cjcb.2019.12.0012