Knockout of miR-184 in zebrafish leads to ocular abnormalities by elevating p21 levels.

miR-184 is one of the most abundant miRNAs expressed in the lens and corneal tissue. Mutations in the seed region of miR-184 are responsible for inherited anterior segment dysgenesis. Animal models recapitulating miR-184-related anterior segment dysgenesis are still lacking, and the molecular basis of ocular abnormalities caused by miR-184 dysfunction has not been well elucidated in vivo. In the present study, we constructed a miR-184-/- zebrafish line by destroying both two dre-mir-184 paralogs with CRISPR-Cas9 technology. Although there were no gross developmental defects, the miR-184-/- zebrafish displayed microphthalmia and cataract phenotypes. Cytoskeletal abnormalities, aggregation of γ-crystallin, and lens fibrosis were induced in miR-184-/- lenses. However, no obvious corneal abnormalities were observed in miR-184-/- zebrafish. Instead of apoptosis, deficiency of miR-184 led to aberrant cell proliferation and a robust increase in p21 levels in zebrafish eyes. Inhibition of p21 by UC2288 compromised the elevation of lens fibrosis markers in miR-184-/- lenses. RNA-seq demonstrated that levels of four transcriptional factors HSF4, Sox9a, CTCF, and Smad6a, all of which could suppress p21 expression, were reduced in miR-184-/- eyes. The predicted zebrafish miR-184 direct target genes (e.g., atp1a3a and nck2a) were identified and verified in miR-184-/- eye tissues. The miR-184-/- zebrafish is the first animal model mimicking miR-184-related anterior segment dysgenesis and could broaden our understanding of the roles of miR-184 in eye development.

Extracellular HSP90 promotes differentiation of lens epithelial cells to fiber cells by activating LRP1-YAP-PROX1 axis.

Capsular residual lens epithelial cells (CRLEC) undergo differentiation to fiber cells for lens regeneration or tansdifferentiation to myofibroblasts leading to posterior capsular opacification (PCO) after cataract surgery. The underlying regulatory mechanism remains unclear. Using human lens epithelial cell lines and the ex vivo cultured rat lens capsular bag model, we found that the lens epithelial cells secrete HSP90α extracellularly (eHSP90) through an autophagy-associated pathway. Administration of recombinant GST-HSP90α protein or its M-domain induces the elongation of rat CRLEC cells with concomitant upregulation of the crucial fiber cell transcriptional factor PROX1and its downstream targets, ß- and γ-crystallins and structure proteins. This regulation is abolished by PROX1 siRNA. GST-HSP90α upregulates PROX1 by binding to LRP1 and activating LRP1-AKT mediated YAP degradation. The upregulation of GST-HSP90α on PROX1 expression and CRLEC cell elongation is inhibited by LRP1 and AKT inhibitors, but activated by YAP-1 inhibitor (VP). These data demonstrated that the capsular residue epithelial cells upregulate and secrete eHSP90α, which in turn drive the differentiation of lens epithelial cell to fiber cells. The recombinant HSP90α protein is a potential novel differentiation regulator during lens regeneration.

Retinitis pigmentosa 2 pathogenic mutants degrade through BAG6/HUWE1 complex.

Retinitis pigmentosa (RP) is the most common inherited retinal degenerative disease which is the major cause of vision loss. X-linked RP patients account for 5%-15% of all inherited RP cases and mutations in RP2 (Retinitis pigmentosa 2) were responsible for about 20% X-linked RP families. A majority of RP2 pathogenic mutations displayed a vulnerable protein stability and degraded rapidly through ubiquitin-proteasome system (UPS). Though the RP2 protein could be readily recovered by proteasome inhibitors, e.g., MG132, their applications for RP2-related RP therapy were limited by their nonspecific characterization. In the present study, we aimed to identify UPS-related factors, such as E3 ligases, which are specifically involved in degradation of RP2 pathogenic mutants. We identified several E3 ligases, such as HUWE1, and the co-chaperon BAG6 specifically interacting with RP2 pathogenic mutants. Knockdown of HUWE1 and BAG6 could partially rescue the reduced protein levels of RP2 mutants. BAG6 is required for recruitment of HUWE1 to ubiquitinate RP2 mutants at the K268 site. The HUWE1 inhibitor BI8622 could restore the levels of RP2 mutant and then the binding to its partner ARL3 in retina cell lines. This study revealed the details of UPS-related degradation of RP2 mutants and possibly provided a potential treatment for RP2-related RP.

The engineered expression of secreted HSPB5-Fc in CHO cells exhibits cytoprotection in vitro.

BACKGROUND: HSPB5 is an ATP-independent molecular chaperone that is induced by heat shock or other proteotoxic stresses. HSPB5 is cytoprotective against stress both intracellularly and extracellularly. It acts as a potential therapeutic candidate in ischemia-reperfusion and neurodegenerative diseases. RESULTS: In this paper, we constructed a recombinant plasmid that expresses and extracellularly secrets a HSPB5-Fc fusion protein (sHSPB5-Fc) at 0.42 µg/ml in CHO-K1 cells. This sHSPB5-Fc protein contains a Fc-tag at the C-terminal extension of HSPB5, facilitating protein-affinity purification. Our study shows that sHSPB5-Fc inhibits heat-induced aggregation of citrate synthase in a time and dose dependent manner in vitro. Administration of sHSPB5-Fc protects lens epithelial cells against cisplatin- or UVB-induced cell apoptosis. It also decreases GFP-Httex1-Q74 insolubility, and reduces the size and cytotoxicity of GFP-Httex1-Q74 aggregates in PC-12 cells. CONCLUSION: This recombinant sHSPB5-Fc exhibits chaperone activity to protect cells against proteotoxicity.

HSP90 as a novel therapeutic target for posterior capsule opacification.

Posterior capsule opacification (PCO) is a common complication of cataract surgery, resulting from a combination of proliferation, migration, epithelial-mesenchymal transition (EMT) of residual capsular epithelial cells and fibrosis of myofibroblasts. HSP90 is known to regulate the proteostasis of cells under pathophysiological conditions. The role of HSP90 in PCO formation, however, is not clear. To do this, the lens epithelial cell lines and an ex vivo cultured rat capsular bag model were used to study the role of HSP90 in PCO formation. The expression of protein and mRNA was measured by immunoblotting and quantitative RT-PCR, and cell apoptosis was measured by TUNEL(TdT-mediated dUTP nick-end labeling). The cell proliferation was measured by cell viability assays. The results showed that 17-AAG (Tanespimycin), an inhibitor of HSP90, suppresses the proliferation of immortalized lens epithelial cell lines HLE-B3, SRA01/04, and mLEC, with IC50 values of 0.27, 0.27, and 0.49 µM, respectively. In an ex vivo cultured rat capsular model, the capsular residual epithelial cells resisted the stress of the capsulorhexis surgery and took 3-6 days to completely overlay the capsular posterior wall. During this process, heat shock factor 1 and its downstream targets HSP90, HSP25, αB-crystallin, and HSP40 were upregulated. Treatment with 17-AAG inhibited the viability of capsular residual epithelial cells and induced the cells apoptosis, characterized by increases in ROS levels, apoptotic DNA injury, and the activation of caspases 9 and 3. HSP90 participated in regulating both EGF receptor (EGFR) and TGF receptor (TGFR) signaling pathways. HSP90 was found to interact with the EGFR, such that inhibition of HSP90 by 17-AAG destabilized the EGFR protein and suppressed p-ERK1/2 and p-AKT levels. 17-AAG also inhibited the TGF-ß-induced phosphorylation of SMAD2/3 and ERK1/2 and the decrease in E-cadherin and ZO-1 expression. Accordingly, these data suggest that the induction of HSP90 protects capsular residual epithelial cells against capsulorhexis-induced stress and participates in regulating the processes of proliferation, EMT and migration of rat capsular residual epithelial cells, at least partly, through the EGFR and TGFR signaling pathways. Treatment with 17-AAG suppresses PCO formation and is therefore a potential therapeutic candidate for PCO prevention.

Hsf4 counteracts Hsf1 transcription activities and increases lens epithelial cell survival in vitro.

The interplay between Hsf4 and Hsf1 plays an important role in the regulation of lens homeostasis. However, the mechanism of the intermolecular association involved is still unclear. In this paper, we find that reconstitution of Hsf4b into Hsf4-/- lens epithelial (mLEC/Hsf4-/-) cells can simultaneously downregulate Hsp70 expression and upregulate the expression of small heat shock proteins Hsp25 and αB-crystallin at both RNA and protein levels. ChIP assay results indicate Hsf4b, which binds to the promoters of Hsp90α, Hsp70.3, Hsp25 and αB-crystallin but not Hsp70.1, can inhibit Hsf1 binding to Hsp70.3 promoter and the heat shock mediated Hsp70 promoter activity by reducing Hsf1 protein expression. Hsf4b N-terminal hydrophobic region can interact with Hsf1 N-terminal hydrophobic region. Their interaction impairs Hsf1's intramolecular interaction between the N- and C-terminal hydrophobic regions, leading to Hsf1's cytosolic retention and protein degradation. Both lysosome inhibitors (chloroquine, pepstatin A plus E64d) and proteasome inhibitor MG132 can inhibit Hsf4-mediated Hsf1 protein degradation, but MG132 can induce Hsf1 activation as well. Upregulation of Hsf4b can significantly inhibit cisplatin and staurosporine induced lens epithelial cell apoptosis through direct upregulation of Hsp25 and αB-crystallin expression. Taken together, our results imply that upregulation of Hsf4b modulates the expression pattern of heat shock proteins in lens tissue by either directly binding to their promoters or promoting Hsf1 protein degradation. Moreover, upregulation of Hsf4b protects lens cell survival by upregulating anti-apoptotic pathways. These studies reveal a novel regulatory mechanism between Hsf1 and Hsf4b in modulating lens epithelial cell homeostasis.

HSF4 promotes G1/S arrest in human lens epithelial cells by stabilizing p53.

The differentiation from constantly dividing epithelial cells into secondary fiber cells is a key step during lens development. Failure in this process, which requires cell proliferation inhibition and cell cycle exit, causes cataract formation. HSF4 (Heat Shock Transcription Factor 4) gene mutations may lead to both congenital and senile cataract. However, how HSF4 mutations induce cataract formation remains obscure. In this study, we demonstrate that HSF4 can suppress the proliferation of human lens epithelial cells (HLECs) by promoting G1/S arrest in a p53-dependent manner. In contrast, HSF4 with cataract causative mutations fail to cause cell cycle arrest and have no obvious effect on cell proliferation. We further identify that HSF4 recruits p53 in the nucleus and promotes its transcriptional activity, leading to the expression of its target gene p21 in HLECs. HSF4, but not its cataract-causing mutants, stabilizes p53 protein and inhibits its ubiquitin degradation. Our data reveal that HSF4 may work as a switch between lens epithelial cell proliferation and secondary fiber cell differentiation, a process which mainly depends on p53. Through demonstration of this novel downstream pathway of HSF4, our results help uncover the pathogenic mechanisms caused by HSF4 mutations.

CERKL interacts with mitochondrial TRX2 and protects retinal cells from oxidative stress-induced apoptosis.

Mutations in the ceramide kinase-like gene (CERKL) are associated with severe retinal degeneration. However, the exact function of the encoded protein (CERKL) remains unknown. Here we show that CERKL interacts with mitochondrial thioredoxin 2 (TRX2) and maintains TRX2 in the reduced redox state. Overexpression of CERKL protects cells from apoptosis under oxidative stress, whereas suppressing CERKL renders cells more sensitive to oxidative stress. In zebrafish, CERKL protein prominently locates in the outer segment and inner segment of the photoreceptor of the retina. Knockdown of CERKL in the zebrafish leads to an increase of retinal cell death, including cone and rod photoreceptor degeneration. Signs of oxidative damage to macromolecules were also detected in CERKL deficient zebrafish retina. Our results show that CERKL interacts with TRX2 and plays a novel key role in the regulation of the TRX2 antioxidant pathway and, for the first time, provides an explanation of how mutations in CERKL may lead to retinal cell death.

Regulation of Hsf4b nuclear translocation and transcription activity by phosphorylation at threonine 472.

Hsf4b, a key regulator of postnatal lens development, is subjected to posttranslational modifications including phosphorylation. However, the phosphorylation sites in Hsf4b and their biological effects on the transcription activity of Hsf4b are poorly understood. Here we examined 17 potential phosphorylation residues in Hsf4b with alanine-scanning assays and found that a T472A mutation diminished Hsf4b-mediated expression of Hsp25 and alphaB-crystallin. In contrast, the phosphomimetic mutation of T472D enhanced their expression. Further investigation demonstrated that Hsf4b could interact with nuclear-transporter importin beta-1 and Hsc70 via amino acids 246-320 and 320-493, respectively. T472A mutation reduced Hsf4bs interaction with importin beta-1, while enhancing its interaction with Hsc7O, resulting in Hsf4b cytosolic re-localization, protein instability and transcription activity attenuation. At the upstream, MEK6 was found to interact with Hsf4b and enhance Hsf4b's nuclear translocation and transcription activity, probably by phosphorylation at sites such as T472. Taken together, our results suggest that phosphotylation of Hsf4b at T472 by protein kinases such as MEI(6 regulates Hsf4b interaction with the importin V I -Hsc7O complex, resulting in blockade of its nuclear translocation and transcriptional activity of Hsf4b.

Glucose regulates heat shock factor 1 transcription activity via mTOR pathway in HCC cell lines.

HSF1-mediated heat shock response is activated in most tumors and plays important roles in regulating tumor homeostasis. However, the signals underlying HSF1 activation is still not completely understood. In this paper, we find that glucose, the dominant tumor energy supplement, participates in regulating HSF1's activation in HCC cell lines. The immunoblotting results indicate that the phosphorylation of HSF1/S326, a hallmark of HSF1 activation, varies between the HCC cell lines (e.g., SMMC7721, HapG2, plc/prf5, and Chang-liver). Glucose, but not 2D-glucose, can induce the phosphorylation of HSF1 at S326 and upregulate the expression of HSF1's downstream alpha B-crystallin and Hsp70 as well as the none-heat shock proteins CSK2 and RBM23 in two tested hepatocellular carcinoma cell lines (prl/prf5 and SMMC7721). Rapamycin, an inhibitor of mTOR, can suppress the glucose-induced phosphorylation of HSF1/S326 and the expression of alpha B-crystallin. Knockdown of HSF1 with shRNA enhances the glucose-depletion-mediated inhibition of plc/prf5 cell proliferation. Our data reveal that HSF1 can be activated by glucose-mTOR pathway, providing an alternative pathway for targeting HSF1 in tumor therapy.

HSF4 regulates DLAD expression and promotes lens de-nucleation.

HSF4 mutations lead to both congenital and age-related cataract. The purpose of this study was to explore the mechanism of cataract formation caused by HSF4 mutations. The degradation of nuclear DNA is essential for the lens fiber differentiation. DNase 2ß (DLAD) is highly expressed in lens cells, and mice with deficiencies in the DLAD gene develop nuclear cataracts. In this study, we found that HSF4 promoted the expression and DNase activity of DLAD by directly binding to the DLAD promoter. In contrast, HSF4 cataract causative mutations failed to bind to the DLAD promoter, abrogating the expression and DNase activity of DLAD. These results were confirmed by HSF4 knockdown in zebrafish, which led to incomplete de-nucleation of the lens and decreased expression and activity of DLAD. Together, our results suggest that HSF4 exerts its function on lens differentiation via positive regulation of DLAD expression and activity, thus facilitating de-nucleation of lens fiber cells. Our demonstration that HSF4 cataract causative mutations abrogate the induction of DLAD expression reveals a novel molecular mechanism regarding how HSF4 mutations cause cataractogenesis.

HSF4 is involved in DNA damage repair through regulation of Rad51.

Heat shock factor protein 4 (HSF4) is expressed exclusively in the ocular lens and plays a critical role in the lens formation and differentiation. Mutations in the HSF4 gene lead to congenital and senile cataract. However, the molecular mechanisms causing this disease have not been well characterized. DNA damage in lens is a crucial risk factor in senile cataract formation, and its timely repair is essential for maintaining the lens' transparency. Our study firstly found evidence that HSF4 contributes to the repair of DNA strand breaks. Yet, this does not occur with cataract causative mutations in HSF4. We verify that DNA damage repair is mediated by the binding of HSF4 to a heat shock element in the Rad51 promoter, a gene which assists in the homologous recombination (HR) repair of DNA strand breaks. HSF4 up-regulates Rad51 expression while mutations in HSF4 fail, and DNA does not get repaired. Camptothecin, which interrupts the regulation of Rad51 by HSF4, also affects DNA damage repair. Additionally, with HSF4 knockdown in the lens of Zebrafish, DNA damage was observed and the protein level of Rad51 was significantly lower. Our study presents the first evidence demonstrating that HSF4 plays a role in DNA damage repair and may contribute a better understanding of congenital cataract formation.

HSF4 Transcriptionally Activates Autophagy by Regulating ATG9a During Lens Terminal Differentiation.

Purpose: HSF4 mutations are responsible for congenital cataract formation. Dysfunction of HSF4 leads to defects in lens terminal differentiation. We aimed to study the mechanism of how HSF4 promotes organelle degradation during lens differentiation. Methods: HSF4del42 mutant mice that developed congenital cataracts were employed. The organelle degradation and autophagic function in lens fibers were detected by immunofluorescence and Immunoblotting. Transcriptome analysis was performed to investigate the differentially expressed genes in HSF4del42 lenses, whereas luciferase report assay and ChIP assay were used to confirm the directly transcriptional regulation of ATG9a by HSF4. Results: HSF4del42 mice displayed delayed organelle clearance and impaired autophagic degradation function in lens fibers. Activation of autophagy by rapamycin ameliorated the defects in organelle clearance in HSF4del42 lenses ex vivo and in vivo. Depletion of HSF4 attenuated autophagic flux by disrupting autophagosome biogenesis and maturation in lens epithelial cells. HSF4 directly transcriptionally activated the core autophagy protein ATG9a. Instead of the canonical ATG9a isoform, the ATG9a-X2 isoform was predominantly expressed in the lens and alleviated autophagic defects in HSF4 KO lens epithelial cells. The ATG9a-X2 protein displayed a short half-life, and rapamycin treatment restored its levels in HSF4 KO lens epithelial cells and HSF4del42 lenses. Conclusions: Our findings demonstrate that HSF4 facilitates organelle degradation probably by transcriptionally activating autophagy during lens terminal differentiation. We first report the involvement of HSF4 in autophagy and the tissue specific splicing of ATG9a. Our study indicates that autophagy activation is a possible therapeutic strategy for HSF4-related congenital cataracts.

Knockout of DLIC1 leads to retinal cone degeneration via disturbing Rab8 transport in zebrafish.

Retinal photoreceptors execute phototransduction functions and require an efficient system for the transport of materials (e.g. proteins and lipids) from inner segments to outer segments. Cytoplasmic dynein 1 is a minus-end-directed microtubule motor and participates in cargo transport in the cytoplasm. However, the roles of dynein 1 motor in photoreceptor cargo transport and retinal development are still ambiguous. In our present study, the light intermediate chain protein DLIC1 (encoded by dync1li1), links activating adaptors to bind diverse cargos in the dynein 1 motor, was depleted using CRISPR-Cas9 technology in zebrafish. The dync1li1-/- zebrafish displayed progressive degeneration of retinal cone photoreceptors, especially blue cones. The retinal rods were not affected in dync1li1-/- zebrafish. Knockout of DLIC1 resulted in abnormal expression and localization of cone opsins in dync1li1-/- retinas. TUNEL staining suggested that apoptosis was induced after aberrant accumulation of cone opsins in photoreceptors of dync1li1-/- zebrafish. Instead of Rab11 transport, Rab8 transport was disturbed in dync1li1-/- retinas. Our data demonstrate that DLIC1 is required for function maintenance and survival of cone photoreceptors, and hint at an essential role of the cytoplasmic dynein 1 motor in photoreceptor cargo transport.

The Therapeutic Roles of Recombinant Hsp90α on Cornea Epithelial Injury.

Purpose: The purpose of this study was to explore the therapeutic role of heat shock protein 90 (Hsp90) in wound healing of injury cornea epithelium. Methods: The right eye of C57BL/6N male mice were performed the debridement wounds in the center of the cornea using an algerbrush II blade. The injured area was determined by staining the cornea with fluorescein sodium and measured with image-J. Immunoblotting, ELISA and immunochemistry were used for determining protein expression. The quantitation PCR was performed to measure mRNA expression. Results: Hsp90α is upregulated at both the mRNA and protein levels, and is secreted extracellularly into the corneal stroma and tear film during the healing process after corneal injury in mice. This upregulation is associated with activation of HSF1. Administration of recombinant exogenous Hsp90α (eHsp90α) speeds up wound healing of injured corneal epithelium. The eHsp90α binds to low-density lipoprotein (LDL)-related protein-1 (LRP-1) on the corneal epithelial cells and increases phosphorylation of AKT at S473, which is associated with proliferation and migration corneal epithelial cells in vitro or vivo. Inhibition of AKT by its inhibitor LY294002 abolishes eHsp90α-induced migration and proliferation of corneal epithelial cells. Conclusion: Hsp90α is upregulated and secreted after corneal injury and acts to promote the healing process. Recombinant Hsp90α may be a promising therapeutic drug candidate for corneal injury.

Biotin attenuates heat shock factor 4b transcriptional activity by lysine 444 biotinylation.

Genetic mutations in HSF4 cause congenital cataracts. HSF4 exhibits both positive and negative regulation on the transcription of heat shock and non-heat shock proteins during lens development, and its activity is regulated by posttranslational modifications. Biotin is an essential vitamin that regulates gene expression through protein biotinylation. In this paper, we report that HSF4b is negatively regulated by biotinylation. Administration of biotin or ectopic bacterial biotin ligase BirA increases HSF4b biotinylation at its C-terminal amino acids from 196 to 493. This attenuates the HSF4b-controlled expression of αB-crystallin in both lens epithelial cells and tested HEK293T cells. HSF4b interacts with holocarboxylase synthetase (HCS), a ubiquitous enzyme for catalyzing protein biotinylation in mammal. Ectopic HA-HCS expression downregulates HSF4b-controlled αB-crystallin expression. Lysine-mutation analyses indicate that HSF4b/K444 is a potential biotinylation site. Mutation K444R reduces the co-precipitation of HSF4b by streptavidin beads and biotin-induced reduction of αB-crystallin expression. Mutations of other lysine residues such as K207R/K209R, K225R, K288R, K294R and K355R in HSF4's C-terminal region do not affect HSF4's expression level and the interaction with streptavidin, but they exhibit distinct regulation on αB-crystallin expression through different mechanisms. HSF4/K294R leads to upregulation of αB-crystallin expression, while mutations K207R/K209R, K225R, K288R, K255R and K435R attenuate HSF4's regulation on αB-crystallin expression. K207R/K209R blocks HSF4 nuclear translocation, and K345R causes HSF4 destabilization. Taken together, the data reveal that biotin maybe a novel factor in modulating HSF4 activity through biotinylation.

Deficiency of heat shock factor 4 promotes lens epithelial cell senescence through upregulating p21cip1 expression.

Genetic mutations in heat shock factor 4 (Hsf4) is associated with both congenital and age-related cataracts. Hsf4 regulates lens development through its ability to both activate and inhibit transcription. Previous studies suggested Hsf4 is involved in modulating cellular senescence depending on p21cip1 and p27 kip1 expression in MEF cells. Here, we found that Hsf4 acts as a suppressor of p21cip1 expression and plays an anti-senescence role during lens development. Knocking out Hsf4 facilitated UVB-induced cellular senescence in mouse lens epithelial cells (mLECs). p21cip1 was upregulated at both the mRNA and protein levels in HSF4-/- mLECs under control and UVB-treated conditions, and knockdown of p21cip1 by siRNA alleviated UVB-induced cellular senescence. HSF4 directly bound to the p21cip1 promoter and increased H3K27m3 levels at the p21cip1 proximal promoter region by recruiting the methyltransferase EZH2. In animal models, p21cip1 was gradually upregulated in wild-type mouse lenses with increasing age, while Hsf4 levels decreased. We generated a Hsf4 mutant mice line (Hsf4del-42) which displayed obvious congenital cataract phenotype. The expression of p21cip1 and senescence-associated cytokines were induced in the cataractous lenses of Hsf4del-42 mice. H3K27m3 and EZH2 levels decreased in p21cip1 promoters in the lenses of Hsf4del-42 mice. The SA-ß-Gal activities were positive in lens epithelia of aged Hsf4null zebrafish compared to wild-type lenses. p21cip1 and senescence-associated cytokines levels were also upregulated in lenses of Hsf4null zebrafish. Accordingly, we propose that HSF4 plays a protective role in lens epithelial cells against cellular senescence during lens development and aging, partly by fine-tuning p21cip1 expression.

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

BACKGROUND: Germline 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. METHODS: HSF4-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. RESULTS: HSF4 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. CONCLUSION: HSF4 regulates lysosomal acidification by controlling the association of αB-crystallin with ATP6V1A and mTOR and regulating ATP6V1A protein stabilization. GENERAL SIGNIFICANCE: This 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.

Knockout of DNase1l1l abrogates lens denucleation process and causes cataract in zebrafish.

Removal of nuclei in lens fiber cells is required for organelle-free zone (OFZ) formation during lens development. Defect in degradation of nuclear DNA leads to cataract formation. DNase2ß degrades nuclear DNA of lens fiber cells during lens differentiation in mouse. Hsf4 is the principal heat shock transcription factor in lens and facilitates the lens differentiation. Knockout of Hsf4 in mouse and zebrafish resulted in lens developmental defect that was characterized by retaining of nuclei in lens fiber cells. In previous in vitro studies, we found that Hsf4 promoted DNase2ß expression in human and mouse lens epithelial cells. In this study, it was found that, instead of DNase2ß, DNase1l1l is uniquely expressed in zebrafish lens and was absent in Hsf4-/- zebrafish lens. Using CRISPR-Cas9 technology, a DNase1l1l knockout zebrafish line was constructed, which developed cataract. Deletion of DNase1l1l totally abrogated lens primary and secondary fiber cell denucleation process, whereas had little effect on the clearance of other organelles. The transcriptional regulation of DNase1l1l was dramatically impaired in Hsf4-/- zebrafish lens. Rescue of DNase1l1l mRNA into Hsf4-/- zebrafish embryos alleviated its defect in lens fiber cell denucleation. Our results in vivo demonstrated that DNase1l1l is the primary DNase responsible for nuclear DNA degradation in lens fiber cells, and Hsf4 can transcriptionally activate DNase1l1l expression in zebrafish.

Blocking the death checkpoint protein TRAIL improves cardiac function after myocardial infarction in monkeys, pigs, and rats.

Myocardial infarction (MI) is a leading cause of death worldwide for which there is no cure. Although cardiac cell death is a well-recognized pathological mechanism of MI, therapeutic blockade of cell death to treat MI is not straightforward. Death receptor 5 (DR5) and its ligand TRAIL [tumor necrosis factor (TNF)-related apoptosis-inducing ligand] are up-regulated in MI, but their roles in pathological remodeling are unknown. Here, we report that blocking TRAIL with a soluble DR5 immunoglobulin fusion protein diminished MI by preventing cardiac cell death and inflammation in rats, pigs, and monkeys. Mechanistically, TRAIL induced the death of cardiomyocytes and recruited and activated leukocytes, directly and indirectly causing cardiac injury. Transcriptome profiling revealed increased expression of inflammatory cytokines in infarcted heart tissue, which was markedly reduced by TRAIL blockade. Together, our findings indicate that TRAIL mediates MI directly by targeting cardiomyocytes and indirectly by affecting myeloid cells, supporting TRAIL blockade as a potential therapeutic strategy for treating MI.