| The mature lens is a transparent organ without blood vessels.The lens has a unique structure,composed of two kinds of cells,one is the lens epithelial cells wrapped in the outer layer.The other is the lens fiber cells that are closely arranged inside,accounting for 99% of the lens cells.In order to establish and maintain the transparency of the lens,the fiber cells are filled with a high concentration of lens protein,and the central fiber cells programmatically remove the nucleus,Golgi apparatus,endoplasmic reticulum,mitochondria and other organelles to form an organelle-free zone(OFZ).If the removal of organelles in lens fiber cells is abnormal,it will lead to cataract.It is of great significance to elucidate the process of lens development and differentiation for understanding the pathogenesis of congenital cataract.HSF4 is a member of the heat shock transcription factor family that is predominantly expressed in the lens.Both Hsf4 deficient mice and zebrafish have cataract.The degradation of organelles such as nucleus,mitochondria and endoplasmic reticulum in the central secondary fibers of the lens was supressed.In mouse and human lens epithelial cells,HSF4 promotes the denucleation process of lens fiber cells by regulating the expression level of DNase2β and DNase activity.The molecular mechanism by which HSF4 promotes the degradation of other organelles is still unclear.Autophagy is a way to degrade organelles.Several studies have shown that autophagy is involved in the lens differentiation process.Mutations in the autophagosome transporter FYCO1 lead to congenital cataracts,and EPG5 knockout mice also have congenital cataract.However,some studies have also shown that the terminal differentiation of the lens is independent of autophagy.And ATG5 is a key protein involved in autophagosome elongation.The lens organelles elimination of ATG5 knockout mice are normal.The mutation of Pik3c3 or Vps34 did not affect organelles degradation during the terminal differentiation of mouse lens fiber.Our previous studies have shown that Hsf4 mutant mice have a severe cataract phenotype and abnormal organelle degradation in lens fiber cells.Disruption of Hsf4 in lens leads to abnormal autophagy function.Rapamycin treatment can active autophagy and accelerate the elimination of organelles in the lens of Hsf4 mutant mice.The generation and fusion of autophagic vesicles are abnormal in lens epithelial cell lacking Hsf4.These results suggest that HSF4 promotes the degradation of organelles in lens fiber cells by mediating autophagy,but the specific molecular mechanism is unclear.This paper further determined the molecular mechanism of HSF4 regulating autophagy during lens differentiation and development.We found that the autophagic core protein ATG9 a in the lens of Hsf4 mutant mice was specifically reduced by transcriptome screening.The m RNA and protein levels of ATG9 a in HSF4-deficient mouse lens epithelial cells were significantly reduced.Through dual luciferase reporter assay and Ch IP assay,it was found that HSF4 transcriptionally activated the expression of ATG9 a.In order to confirm the role of ATG9 a in lens differentiation,we found that ATG9 a was specifically expressed in lens differentiating fiber cells by immunofluorescence assay,and ATG9 a co-localized with autophagy marker LC3 and organelles such as mitochondria and endoplasmic reticulum.This suggests that ATG9 a may be involved in the autophagy process and organelle degradation of lens fiber cells during lens differentiation process.And unlike the standard ATG9 a Fl-a transcript,we found a special ATG9 a transcript X3(expressed protein ATG9 a X2)in mouse lens.To determine the function of two transcripts in autophagy,immunofluorescence assay showed that ATG9 a Fl-a was diffusely distributed in the cytoplasm,and ATG9 a X2 formed large dotted fluorescence in the cytoplasm and co-localized with autophagic vesicles.We complemented two transcripts of ATG9 a in the mouse lens epithelial cell lacking Hsf4,and the results showed that ATG9 a could recover the autophagy function of Hsf4 knockout lens epithelial cells.Compared with ATG9 a Fl-a transcript,the autophagy function of Hsf4 knockout lens epithelial cells was significantly recovered after complementing ATG9 a X3.These results suggest that ATG9 a X3 transcripts plays a key role in autophagy during mouse lens differentiation and development.Rapamycin was used to treat the lens of Hsf4 mutant mice and Hsf4 knockout lens epithelial cells in vitro.The results showed that rapamycin could partially restore the protein level of ATG9 a X2.In summary,our results indicate that HSF4 regulates autophagy by transcriptional activation of ATG9 a during lens differentiation,which is of great significance to reveal the mechanism by which HSF4 regulates autophagy to promote organelles degradation in lens differentiation. |
