TGF-β2 increases eHSP90α secretion via upregulating secretory autophagy pathway.

BACKGROUND: Extracellular heat shock protein 90α (eHSP90α) regulates diverse cellular processes such as wound healing, tumor metastasis, angiogenesis and cell differentiation etc. Our previous data show that lens epithelial cells can secret eHSP90α, and administration of eHSP90α exerts a critical regulation for lens regeneration by promoting the differentiation of lens epithelial cells (ECs) to lens fiber cells. Autophagy is proposed to regulate the eHSP90α secretion from lens epithelial cells. However, the regulatory mechanism remains unclear. METHODS: Lens ECs cell line SRA01/04, ex vivo cultured rat lens capsular bags and primary rat lens ECs were used in this study. The immunoblotting and qPCR were used for measuring the expression of proteins and mRNAs. The immunofluorescence staining assay was for testing the autophagosomes or protein co-localization. siRNA or CRISPR-Cas9 were used to knock down genes' expression in cells in vitro. The ChIP assay was used to study the interaction of transcriptional factor to the promoter of target genes. The nuclear and cytoplasmic extraction assay was for testing the nuclear translocation of TFEB and TFE3. RESULTS: eHSP90α is secreted through the secretory autophagy pathway in lens epithelial cells in vitro or in the residual anterior ECs of capsular bags ex vivo. HSP90α interacts with adaptor protein TRIM16, and was recruited to the R-SNARE protein SEC22B on the surface of secretory autophagosome membrane for secretion. Silencing autophagic ATG7 or components of secretory autophagy pathways, such as TRIM16, SEC22B and Q-SNARE STX3, STX4 and SNAP23 downregulates HSP90α secretion. TGF-β2 upregulates the eHSP90α secretion via upregulating secretory autophagy pathway. TGF-β2 treatment results in the upregulation of the expression of ATG7, Beclin1, LC3B at both protein and mRNA levels through activating p38 and ERK1/2. In addition, TGF-β2 increases SEC22B expression through ERK1/2-induced expression and nuclear translocation of transcriptional factors TFEB and TFE3, which form the heterodimer binding to the promoter of SEC22B. Functionally, the upregulated eHSP90α can trigger TGF-β2-induced differentiation of lens ECs to fiber cells. CONCLUSIONS: In lens cells, the eHSP90α's secretion undertakes secretory autophagy pathway through interacting with TRIM16 and SEC22B. TGF-β2 elevates eHSP90α secretion through upregulating secretory autophagy pathway, which in turn promote TGF-β2-induced lens epithelial cells' differentiation to lens fiber cells.