Heat shock factor 4 regulates lens epithelial cell homeostasis by working with lysosome and anti-apoptosis pathways

Activation of Heat shock factor 4-mediated heat shock response is closely associated with postnatal lens development. HSF4 controls the expression of small heat shock proteins (e.g. HSP25 and CRYAB) in lens epithelial cells. However, their roles in modulating lens epithelium homeostasis remain unclear. In this paper, we find that HSF4 is developmentally expressed in mouse lens epithelium and fiber tissue. HSF4 and alpha B-crystallin can selectively protect lens epithelial cells from cisplatin and H2O2 induced apoptosis by stabilizing mitochondrial membrane potential (ΔY_m) and reducing ROS production. In addition, to our surprise, HSF4 is involved in upregulating lysosome activity. We found mLEC/HA-Hsf4 cells to have increased DLAD expression, lysosome acidity, cathepsin B activity, and degradation of plasmid DNA and GFP-LC3 protein when compared to mLEC/Hsf4-/- cells. Knocking down Cryab from mLEC/HA-Hsf4 cells inhibits HSF4-mediated lysosome acidification, while overexpression of CRYAB can upregulate cathepsin B activity in mLEC/Hsf4-/- cells. CRAYAB can interact with ATP6V1/A the A subunit of the H⁺ pump vacuolar ATPase, and is colocalized to lamp1 and lamp2 in the lysosome. Collectively, these results suggest that in addition to modulating anti-apoptosis, HSF4 is able to regulate lysosome activity by at least controlling alpha B-crystallin expression, shedding light on a novel molecular mechanism of HSF4 in regulating lens epithelial cell homeostasis.     

Heat shock factor 4 regulates lysosome activity by modulating the αB-crystallin-ATP6V1A-mTOR complex in ocular lens

BackgroundGermline mutations in heat shock factor 4 (HSF4) cause congenital cataracts. Previously, we have shown that HSF4 is involved in regulating lysosomal pH in mouse lens epithelial cell in vitro. However, the underlying mechanism remains unclear.MethodsHSF4-deficient mouse lens epithelial cell lines and zebrafish were used in this study. Immunoblotting and quantitative RT-PCR were used for expression analysis. The protein-protein interactions were tested with GST-pull downs. The lysosomes were fractioned by ultracentrifugation.ResultsHSF4 deficiency or knock down of αB-crystallin elevates lysosomal pH and increases the ubiquitination and degradation of ATP6V1A by the proteasome. αB-crystallin localizes partially in the lysosome and interacts solely with the ATP6V1A protein of the V1 complex of V-ATPase. Furthermore, αB-crystallin can co-precipitate with mTORC1 and ATP6V1A in GST pull down assays. Inhibition of mTORC1 by rapamycin or siRNA can lead to dissociation of αB-crystallin from the ATP6V1A and mTORC1complex, shortening the half-life of ATP6V1A and increasing the lysosomal pH. Mutation of ATP6V1A/S441A (the predicted mTOR phosphorylation site) reduces its association with αB-crystallin. In the zebrafish model, HSF4 deficiency reduces αB-crystallin expression and elevates the lysosomal pH in lens tissues.ConclusionHSF4 regulates lysosomal acidification by controlling the association of αB-crystallin with ATP6V1A and mTOR and regulating ATP6V1A protein stabilization.General significanceThis study uncovers a novel function of αB-crystallin, demonstrating that αB-crystallin can regulate lysosomal ATP6V1A protein stabilization by complexing to ATP6V1A and mTOR. This highlights a novel mechanism by which HSF4 regulates the proteolytic process of organelles during lens development.     

Heat shock factor 2 is required for maintaining proteostasis against febrile-range thermal stress and polyglutamine aggregation

Heat shock response is characterized by the induction of heat shock proteins (HSPs), which facilitate protein folding, and non-HSP proteins with diverse functions, including protein degradation, and is regulated by heat shock factors (HSFs). HSF1 is a master regulator of HSP expression during heat shock in mammals, as is HSF3 in avians. HSF2 plays roles in development of the brain and reproductive organs. However, the fundamental roles of HSF2 in vertebrate cells have not been identified. Here we find that vertebrate HSF2 is activated during heat shock in the physiological range. HSF2 deficiency reduces threshold for chicken HSF3 or mouse HSF1 activation, resulting in increased HSP expression during mild heat shock. HSF2-null cells are more sensitive to sustained mild heat shock than wild-type cells, associated with the accumulation of ubiquitylated misfolded proteins. Furthermore, loss of HSF2 function increases the accumulation of aggregated polyglutamine protein and shortens the lifespan of R6/2 Huntington's disease mice, partly through αB-crystallin expression. These results identify HSF2 as a major regulator of proteostasis capacity against febrile-range thermal stress and suggest that HSF2 could be a promising therapeutic target for protein-misfolding diseases.