Regulation Of Hsf4 On Lysosomal Activity

BackgroundDenature or aggregation of ocular lens intracellular proteins blocks the light transmition to the retina causing the cataract. Cataract incidence is about 60-80 woldwide and most of them occur in the age over 50. Cataract is the predominant cause of blindness. About 50% of the blindness is induced by cataract.Under normal circumstances, the lens in peoples’ eyes is transparent, light can pass through it to the retina and people can see clearly the objects. Lens is composed of two kinds of cells, the anterior epithelial cells and bulk of fiber cells. The equator lens epithelial cells form germline proliferation center, which can life-long proliferate and differentiate into the cortial fiber. Lens fibers consist of cortial fiber and the centre terminal differentiated fiber cells, which are filled with bulk of crystalline proteins and depletion of the membrane organelles e.g. mitochondria, endoplasm reticulum, Golgi, and nucleus forming the transparent center. This center is also named as organelle free center. It is unclear how the nuclear depletion is regulated during the lens fiber cell differentiation. Some evidence suggested that autophage-lysosome and ubiquitin-proteasome play important roles in organelle-removing. However, the molecular mechanism is unclear.Hsf4 is a family member of heat shock transcription factors that have the conserved HSE binding domain and heat shock regulator domains. These family members can sensitize the variety of stresses signals such as heat shock, H2O2, and proteotoxin and upregulate the expression of heat shock proteins. The latter then protect cells from damage by remodeling the protein refolding, distribution and degradation. Hsf4 dominantly expresses in spleen, ocular lens, cardiomyocyte and brain. Genetic mutation of Hsf4 in DNA binding domain is closely associated with human family autosome chromosome dominant and reseseive cataracts. Hsf4 gene knockout cause the neonatal cataracts with impairment of lens fiber cell differentiation characterizing with defective of fiber cell nuclear depletion. It is reported that Hsf4 control the expression of DNase II beta, a critical enzyme for the degradation of fiber cell nuclear DNA. DNase II beta is expressed in lysosome. Therefore, we hypothesis that Hsf4 may participate in the regulation of fiber cell nuclear degradation by associating with lysosome activity.AimsTo determine Hsf4’ regulatory effects on the lysosome activity by using the mouse lens epithelial cells m LEC/Hsf4-/- and ml EC/Hsf4 b.Methods1.To determine Hsf4’s regulatory roles on autophage by testing the ratio of LC3I/LC3 II and LC3 granulation with immunoblotting and immunohistochemistry staining.2.Testing the degradation of GFP-LC3 speed in the lysosome of m LEC/hsf4-/- and m LEC/Hsf4 in present of the autophagy activator Rapamycin or the lysosome inhibitor Chloroquine) by using the immunoblotting assay.3.Testing the expression and localization of lysosome by immunoblotting and immunohistochemistry staining the expression of LAMP1、LAMP2 in the mlec/Hsf4-/- and m LEC/Hsf4 cells.4.Testing the cathepsin B activity in m LEC/hsf4-/- and m LEC/Hsf4 cells by fluorescent staining with Magic Red to determine the regulatory activity of Hsf4 on lysosome.5. Test the interactions between alpha B-crystallins and ATP6 VIA by CO-immunoprecipitation to investigate whether downstream target of Hsf4, such as alpha B-crystallin is involved in regulating lysosome p H value by regulating the proton bump ATP6 Via.Results1.The overexpression of HSF4 b result in enhanced autophagic response.2.Autophagy in lens epithelial cells could enhance by the treatment with Rapamycin in short time.With the extending of treatmental time, the expression of LC3-Ⅰand LC3-Ⅱin mlec-HA-Hsf4 b significantly decrease, probably due to Hsf4 b promotes lysosomal activity to degrade more LC3-Ⅱ.3.By detecting the generation of free GFP from GFP-LC3, indirectly explain that Hsf4 b can enhance lysosomal activity of epithelial cells. With the treatment with Rapamycin, the activity of lysosome both increase.4.Use different concentrations of chloroquine to treat with lens epithelial cells, by detection of expression of free GFP will reveal that in a certain range of concentration, lysosomal activity is partially inhibited as the improvement of concentration, once more than the concentration, lysosomal activity may be completely suppressed.5.Staining with Magic Red finds that overexpression of Hsf4 b can affect the activity of cathepsin B, directly showing that Hsf4 b increase lysosomal activity.6.The detection of LAMP1、LAMP2 in lens epithelial cells by Western blot finds that Hsf4 b has no influence on the number of lysosome, but on the distributions. LAMP1 usually distributes around the nucleus in the state of accumulation in mlec cell, while is evenly distributed across the nucleus of 4b cell; LAMP2 is evenly distributed across the nucleus of mlec cell, but found in cytoplasm and nucleus of 4b cell.7. Have no found interactions with crystallinsαB that is Hsf4b-activated gene and ATP6 VIA by CO-IP, it still continue to further study.Conclusions1. Hsf4 is involved in regulating autophagy activity.2. Hsf4 participates in upregulating lysosome protein-hydrolysis activity, potentially by modulating lysosome’ p H value.