ABSTRACT
The lens is transparent, non-vascular, elastic and wrapped in a transparent capsule. The lens oppacity of AQP5-/- mice was increased more than that of wild-type (AQP5+/+ ) mice. In this study, we explored the potential functional role of circular RNAs (circRNAs) and transcription factor HSF4 in lens opacity in aquaporin 5 (AQP5) knockout (AQP5-/- ) mice. Autophagy was impaired in the lens tissues of AQP5-/- mice. Autophagic lysosomes in lens epithelial cells of AQP5-/- mice were increased compared with AQP5+/+ mice, based on analysis by transmission electron microscopy. The genetic information of the mice lens was obtained by high-throughput sequencing, and then the downstream genes were analysed. A circRNA-miRNA-mRNA network related to lysosomal pathway was constructed by the bioinformatics analysis of the differentially expressed circRNAs. Based on the prediction of the TargetScan website and the validation by dual luciferase reporter assay and RNA immunoprecipitation-qPCR, we found that circRNA (Chr16: 33421321-33468183+) inhibited the function of HSF4 by sponging microRNA (miR-149-5p), and it downregulated the normal expression of lysosome-related mRNAs. The accumulation of autophagic lysosome may be one of the reasons for the abnormal development of the lens in AQP5-/- mice.
Subject(s)
Lens, Crystalline , MicroRNAs , Animals , Mice , RNA, Circular/metabolism , Aquaporin 5/genetics , Aquaporin 5/metabolism , MicroRNAs/genetics , Lens, Crystalline/metabolism , RNA, Messenger/metabolismABSTRACT
Heat shock factors (HSFs) are a family of transcription factors, composed of HSF1, HSF2, and HSF4, to regulate cell stress reaction for maintaining cellular homeostasis in response to adverse stimuli. Recent studies have disclosed the roles of HSF1 and HSF2 in modulating tumor development, including colorectal cancer (CRC). However, HSF4, which is closely associated with pathology of congenital cataracts, remains less studied in tumors. In this study, we aimed to describe the regulatory effects of HSF4 and underlying molecular mechanism in CRC progression. By bioinformatic analysis of TCGA database and TMA-IHC assay, we identified that the expression of HSF4 was significantly upregulated in CRCs compared with normal colonic tissues and was a prognostic factor of poor outcomes of CRC patients. Function assays, including CCK-8, colony formation, transwell assays, and xenografted mouse model, were employed to verify that HSF4 promoted cell growth, colony formation, invasion of CRC cells in vitro, and tumor growth in vivo as a potential oncogenic factor. Mechanistically, results of Chromatin immunoprecipitation (ChIP) and immunoblotting assays revealed that HSF4 associated directly to MET promoter to enhance expression of c-MET, a well-known oncogene in multiple cancers, thus fueling the activity of downstream ERK1/2 and AKT signaling pathways. In further rescue experiments, restoration of c-MET expression abolished inhibitory cell growth and invasion induced by downregulated HSF4 expression. To sum up, our findings describe a crucial role of HSF4 in CRC progression by enhancing activity of c-MET and downstream ERK1/2 and AKT signaling pathways, and highlight HSF4 as a potential therapeutic target for anti-CRC treatment.
Subject(s)
Cataract , Colorectal Neoplasms , Mice , Animals , Heat Shock Transcription Factors/genetics , Proto-Oncogene Proteins c-akt/metabolism , Transcription Factors/metabolism , Colorectal Neoplasms/genetics , Cell Line, Tumor , Cell Proliferation , Gene Expression Regulation, NeoplasticABSTRACT
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.
ABSTRACT
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.
Subject(s)
Cyclin-Dependent Kinase Inhibitor p21/genetics , Heat Shock Transcription Factors/deficiency , Lens, Crystalline/pathology , Zebrafish Proteins/deficiency , Zebrafish Proteins/genetics , Aging/genetics , Animals , Animals, Genetically Modified , Cataract/genetics , Cataract/pathology , Cell Line , Cellular Senescence/genetics , Cellular Senescence/radiation effects , DNA Methylation , Disease Models, Animal , Enhancer of Zeste Homolog 2 Protein/metabolism , Epithelial Cells/pathology , Epithelial Cells/radiation effects , Gene Expression Regulation, Developmental , Gene Knockout Techniques , Heat Shock Transcription Factors/genetics , Histones/genetics , Histones/metabolism , Humans , Lens, Crystalline/cytology , Lens, Crystalline/growth & development , Lens, Crystalline/radiation effects , Mice , Promoter Regions, Genetic , Ultraviolet Rays/adverse effects , Zebrafish , Zebrafish Proteins/metabolismABSTRACT
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.
Subject(s)
Cataract/genetics , Deoxyribonucleases/genetics , Gene Expression Regulation, Developmental , Heat Shock Transcription Factors/metabolism , Lens, Crystalline/embryology , Zebrafish Proteins/genetics , Animals , Animals, Genetically Modified , CRISPR-Cas Systems/genetics , Cataract/pathology , Cell Nucleus/metabolism , Deoxyribonucleases/metabolism , Disease Models, Animal , Embryo, Nonmammalian , Epithelial Cells/cytology , Epithelial Cells/metabolism , Female , Gene Knockout Techniques , Heat Shock Transcription Factors/genetics , Humans , Lens, Crystalline/cytology , Lens, Crystalline/metabolism , Lens, Crystalline/pathology , Male , Zebrafish , Zebrafish Proteins/metabolismABSTRACT
Objective To analysis the pathogenic mutation and the clinical characteristics of a three generation family with congenital pulverulent cataract. Methods A congenital cataract family was chosen from the First Affiliated Hospital of AnHui Medical University,5 ml peripheral blood was obtained from each family member to extract genomic DNA. Next generation sequencing was used to detect the mutation in proband (Ⅱ5),Ⅱ6 and Ⅲ8, and Sanger sequencing was applied to verify pathogenic mutation in the whole family members. The mutation site was compared with the gene sequence of 10000 normal Chinese. PolyPhen-2 and SIFT were applied to analysis the alteration on the protein structure and function and its possible pathogenesis. This study followed the Declaration of Helsinki and was approved by the Ethics Committee of AnHui Medical University ( NO. PJ2017-5-17 ) . All patients signed informed consent. Results The pedigree consisted of 19 members of three generations,including 10 patients and 9 normal family members. Heterozygous mutation of GJA3 gene c. 427G>A ( p. G143R) was detected in all patients of the pedigree,but was not found in normal members of the pedigree and 10000 normal Chinese. The score calculated from SIFT and PolyPhen-2 indicated that the mutation probably had malignant effect on normal protein structure,Swiss-model website analysis showed that the mutation likely altered the secondary structure of the protein CX 46 by reducing anα-helix between 107-115 amino acids. Meanwhile,c. 1325-1G>T mutation of HSF4 gene were detected in Ⅱ5 and Ⅲ8, which was not found in other family members and 10000 normal Chinese. HSF and MaxEntScan results showed that the mutation probably had serious effect on the splicing of mRNA. The cataract development rates of Ⅱ5 andⅢ8 were faster than that of the same age in the same generation of the pedigree,and the morphology of lens opacity was changed. Conclusions The heterozygous c. 427G>A mutation in GJA3 gene is responsible for pulverulent cataract in this family,meanwhile the c. 1325-1G>T mutation in HSF4 gene may change the type of phacoscotasmus and accelerate the progress of disease.
ABSTRACT
Objective@#To analysis the pathogenic mutation and the clinical characteristics of a three generation family with congenital pulverulent cataract.@*Methods@#A congenital cataract family was chosen from the First Affiliated Hospital of AnHui Medical University, 5 ml peripheral blood was obtained from each family member to extract genomic DNA.Next generation sequencing was used to detect the mutation in proband (Ⅱ5), Ⅱ6 and Ⅲ8, and Sanger sequencing was applied to verify pathogenic mutation in the whole family members.The mutation site was compared with the gene sequence of 10 000 normal Chinese.PolyPhen-2 and SIFT were applied to analysis the alteration on the protein structure and function and its possible pathogenesis.This study followed the Declaration of Helsinki and was approved by the Ethics Committee of AnHui Medical University (NO.PJ2017-5-17). All patients signed informed consent.@*Results@#The pedigree consisted of 19 members of three generations, including 10 patients and 9 normal family members.Heterozygous mutation of GJA3 gene c. 427G>A (p.G143R) was detected in all patients of the pedigree, but was not found in normal members of the pedigree and 10 000 normal Chinese.The score calculated from SIFT and PolyPhen-2 indicated that the mutation probably had malignant effect on normal protein structure, Swiss-model website analysis showed that the mutation likely altered the secondary structure of the protein CX 46 by reducing an α-helix between 107-115 amino acids.Meanwhile, c.1325-1G>T mutation of HSF4 gene were detected in Ⅱ5 and Ⅲ8, which was not found in other family members and 10 000 normal Chinese.HSF and MaxEntScan results showed that the mutation probably had serious effect on the splicing of mRNA.The cataract development rates of Ⅱ5 and Ⅲ8 were faster than that of the same age in the same generation of the pedigree, and the morphology of lens opacity was changed.@*Conclusions@#The heterozygous c. 427G>A mutation in GJA3 gene is responsible for pulverulent cataract in this family, meanwhile the c. 1325-1G>T mutation in HSF4 gene may change the type of phacoscotasmus and accelerate the progress of disease.
ABSTRACT
Dysfunction of HSF4 is associated with congenital cataracts. HSF4 transcription activity is turned on and regulated by phosphorylation during early postnatal lens development. Our previous data suggested that mutation HSF4b/S299A can upregulate HSF4 transcription activity in vitro, but the biological significance of posttranslational modification on HSF4/S299 during lens development remains unclear. Here, we found that the mutation HSF4/S299A can upregulate the expression of HSP25 and alpha B-crystallin at both protein and mRNA levels in mouse the lens epithelial cell line, but HSF4/S299D does not. Using the rabbit polyclonal antibody against phospho-S299 of HSF4, we found that EGF and ectopic expression of MEK1 can increase the phosphorylation of HSF4/S299 and induce HSF4 sumoylation, and these effects are inhibited by U0126. ERK1/2 can phosphorylate the S299 in HSF4/wt but not in HSF4/S299A in the in vitro kinase assay. Functionally, ectopic MEK1 can inhibit HSF4-controled alpha B-crystallin expression but has less effect on HSF4/S299A. EGF can upregulate phospho-HSF4/S299 and downregulate alpha B-crystallin expression in P3 mouse lens, and this downregulation is suppressed by U0126. During mouse lens development, phosphorylation of HSF4/S299 is downregulated in P3 lens and upregulated in P7 and P14 lens. However, in 2 months old lens, both phosphorylation of HSF4/S299 and total HSF4 protein are decreased. Interestingly, ERK1/2 activity is lower in P3 lens than in P7 and P14 lens, which is in line with the phosphorylation of HSF4/S299. Taken together, our data demonstrate that HSF4/299 is a phosphorylation target of MEK1-ERK1/2, and phosphorylation of S299 is responsible for tuning down HSF4 transcription activity during postnatal lens development.
Subject(s)
Heat Shock Transcription Factors/genetics , Lens, Crystalline/metabolism , MAP Kinase Signaling System , Amino Acid Substitution , Animals , Cells, Cultured , Down-Regulation , Gene Knockout Techniques , HSP27 Heat-Shock Proteins/genetics , Heat Shock Transcription Factors/chemistry , Heat Shock Transcription Factors/deficiency , Heat Shock Transcription Factors/metabolism , Heat-Shock Proteins/genetics , Humans , Lens, Crystalline/growth & development , Mice , Molecular Chaperones , Neoplasm Proteins/genetics , Phosphorylation , Point Mutation , RNA, Messenger/genetics , RNA, Messenger/metabolism , Serine/metabolism , Transcription, Genetic , Up-Regulation , alpha-Crystallin B Chain/geneticsABSTRACT
BACKGROUND: Congenital cataract, a kind of cataract presenting at birth or during early childhood, is a leading cause of childhood blindness. To date, more than 30 genes on different chromosomes are known to cause this disorder. This study aimed to identify the HSF4 mutations in a cohort from Chinese families affected with congenital cataracts. METHODS: Forty-two unrelated non-syndromic congenital cataract families and 112 ethnically matched controls from southeast China were recruited from the southeast of China. We employed Sanger sequencing method to discover the variants. To confirm the novel mutations, STR haplotypes were constructed to check the co-segregation with congenital cataract. The pathogenic potential of the novel mutations were assessed using bioinformatics tools including SIFT, Polyphen2, and Human Splicing Finder. The pathogenicity of all the mutations was evaluated by the guidelines of American College of Medical Genetics and InterVar software. RESULTS: No previously reported HSF4 mutations were found in all the congenital cataract families. Five novel HSF4 mutations including c.187 T > C (p.Phe63Leu), c.218G > T (p.Arg73Leu), c.233A > G (p.Tyr78Cys), IVS5 c.233-1G > A and c.314G > C (p.Ser105Thr) were identified in five unrelated families with congenital cataracts, respectively. These mutations co-segregated with all affected individuals in each family were not observed in the unaffected family members or in 112 unrelated controls. All five mutations were categorized to be the disease "pathogenic" according to ACMG guidelines and using InterVar software. Mutations in the HSF4 were responsible for 11.90% Chinese families with congenital cataracts in our cohort. CONCLUSIONS: In the study, we identified five novel HSF4 mutations in Chinese families with congenital cataracts. Our results expand the spectrum of HSF4 mutations causing congenital cataracts, which may be helpful for the molecular diagnosis of congenital cataracts in the era of precision medicine.
Subject(s)
Cataract/genetics , Heat Shock Transcription Factors/genetics , Mutation/genetics , Asian People/genetics , China , Cohort Studies , Family , Female , Haplotypes/genetics , Humans , Male , Pedigree , RNA Splicing/geneticsABSTRACT
The major causes for cataract formation are free radicals, which are neutralized by the endogenous antioxidants. However, how the human lens clean these harmful free radicals is still unclear. Transcriptional factor heat shock factor 4 (HSF4) is a cataract-causing gene and plays important roles during lens development. Here we show that HMOX-1, an anti-oxidase, is a bona fide transcriptional target gene of HSF4 in HLECs (human lens epithelial cells). HSF4 directly binds to the HSE element in HMOX-1 promoter to mediate its mRNA transcription and protein accumulation. The HSE element located at the region of -389â¯bp to -362â¯bp upstream from the TSS (transcription start site), which is critical for HMOX-1 transcriptional activation. Furthermore, knockdown of HSF4 by siRNA inhibited HMOX-1 expression. Thus, these data revealed a novel transcription target of HSF4 and provided new insights into anti-oxidation regulation in lens and age-related cataract.
Subject(s)
Epithelial Cells/metabolism , Heat Shock Transcription Factors/physiology , Heme Oxygenase-1/genetics , Lens, Crystalline/metabolism , Animals , Cells, Cultured , Gene Expression Regulation , Heat Shock Transcription Factors/metabolism , Heme Oxygenase-1/metabolism , Humans , Lens, Crystalline/cytology , Promoter Regions, Genetic , Rabbits , Response Elements , Transcriptional ActivationABSTRACT
Heat shock transcription factors (Hsfs) regulate transcription of heat shock proteins as well as other genes whose promoters contain heat shock elements (HSEs). There are at least five Hsfs in mammalian cells, Hsf1, Hsf2, Hsf3, Hsf4, and Hsfy (Wu, Annu Rev Cell Dev Biol 11:441-469, 1995; Morimoto, Genes Dev 12:3788-3796, 1998; Tessari et al., Mol Hum Repord 4:253-258, 2004; Fujimoto et al., Mol Biol Cell 21:106-116, 2010; Nakai et al., Mol Cell Biol 17:469-481, 1997; Sarge et al., Genes Dev 5:1902-1911, 1991). To understand the physiological roles of Hsf1, Hsf2, and Hsf4 in vivo, we generated knockout mouse lines for these factors (Zhang et al., J Cell Biochem 86:376-393, 2002; Wang et al., Genesis 36:48-61, 2003; Min et al., Genesis 40:205-217, 2004). Numbers of other laboratories have also generated Hsf1 (Xiao et al., EMBO J 18:5943-5952, 1999; Sugahara et al., Hear Res 182:88-96, 2003), Hsf2 (McMillan et al., Mol Cell Biol 22:8005-8014, 2002; Kallio et al., EMBO J 21:2591-2601, 2002), and Hsf4 (Fujimoto et al., EMBO J 23:4297-4306, 2004) knockout mouse models. In this chapter, we describe the design of the targeting vectors, the plasmids used, and the successful generation of mice lacking the individual genes. We also briefly describe what we have learned about the physiological functions of these genes in vivo.
Subject(s)
Gene Deletion , Gene Knockout Techniques/methods , Heat Shock Transcription Factors/genetics , Heat-Shock Proteins/genetics , Transcription Factors/genetics , Animals , Genetic Vectors , Male , Mice , Mice, KnockoutABSTRACT
Heat shock factor 4 controls the transcription of small heat shock proteins (e.g., HSP25, alpha B-cyrstallin, and r-crystallin), that play important roles in modulating lens proteostasis. However, the molecular mechanism underlying HSF4-mediated transcription is still unclear. Using yeast two hybrid, we found that HSF4 interacts with the ATP-dependent DEXD/H-box RNA helicase UAP56, and their interaction in lens epithelial cell line was further confirmed by GST-pull down assay. UAP56 is a vital regulator of pre-mRNA splicing and mature mRNA nuclear export. The immunofluorescence assay showed that HSF4 and UBA56 co-localize with each other in the nucleus of lens epithelial cells. Ectopic UAP56 upregulated HSF4-controlled HSP25 and alpha B-crystallin proteins expression, while knocking down UAP56 by shRNA reversed it. Moreover, UAP56 interacts with and facilitates the nuclear exportation of HSP25 and alpha B-crystallin mRNA without impacting their total mRNA expression level. In lens tissues, both UAP56 and HSF4 are expressed in the same nucleus of lens fiber cells, and their expression levels are simultaneously reduced with fiber cell maturation. Taken together, these data suggested that UAP56 is a novel regulator of HSF4 and might upregulate HSF4's downstream mRNA maturation and nuclear exportation.
Subject(s)
DEAD-box RNA Helicases/metabolism , Heat Shock Transcription Factors/metabolism , Heat-Shock Proteins/metabolism , Neoplasm Proteins/metabolism , alpha-Crystallin B Chain/metabolism , Animals , Cell Nucleus/metabolism , Epithelial Cells/metabolism , Heat-Shock Proteins/genetics , Humans , Lens, Crystalline/cytology , Mice , Molecular Chaperones , Neoplasm Proteins/genetics , Protein BindingABSTRACT
Heat shock factor 4 (HSF4) is a member of the HSF family. In this study, by using data from the Cancer Genome Atlas-Colorectal Cancer (TCGA-CRC), we investigated the expression profile and the prognostic value of the HSF4 in terms of overall survival (OS) and recurrence free survival (RFS) in CRC patients. RNA-Seq data showed that HSF4 RNA expression was significantly higher in CRC tissues (N = 380) than in the corresponding normal tissues (N = 51) (mean ± SD: 3.56 ± 1.28 vs. 1.85 ± 0.87, P < 0.0001). High HSF4 expression group had significantly higher ratio of stages III/IV patients (52/86, 60.5%) than low HSF4 expression group (110/264, 41.7%; P = 0.0024). Besides, the high HSF4 expression group also had significantly increased expression of CEA (CEA ≥ 5, 26/51, 51.0% vs. 64/186, 34.4%), higher proportion of recurrence (32/86, 37.2% vs. 48/254, 18.9%, P = 0.0005) and death (36/90, 40.0% vs. 49/277, 17.7%, P < 0.0001) compared with the low HSF4 expression group. Multivariate analysis confirmed that high HSF4 expression was an independent prognostic factor of poor OS (HR = 2.111, 95%CI: 1.350-3.302, P = 0.001) and RFS (HR = 1.958, 95%CI: 1.224-3.131, P = 0.005). Bioinformatic analysis showed that HSF4 can directly interact with DUSP26, ZBED8, and MAPK14. It is also coexpressed with PTGER1, COL11A2, CLPS, and ARSA and colocalized with PTGER1, ADRB1, PEX12, CLPS, PSEN2, KCNJ5, CPA1, ARSA, PNLIP, IRX4, CPA2, IDUA, BCKDHA, and CTRL. We hypothesized that HSF4 might exert its oncogenic effects in CRC via some of these genes. © 2017 IUBMB Life, 69(12):956-961, 2017.
Subject(s)
Biomarkers, Tumor/genetics , Carcinoembryonic Antigen/genetics , Colorectal Neoplasms/diagnosis , Colorectal Neoplasms/mortality , Heat Shock Transcription Factors/genetics , Aged , Biomarkers, Tumor/blood , Carcinoembryonic Antigen/blood , Colorectal Neoplasms/blood , Colorectal Neoplasms/genetics , Computational Biology , Disease-Free Survival , Female , Gene Expression , Gene Regulatory Networks , Heat Shock Transcription Factors/blood , Humans , Kaplan-Meier Estimate , Male , Middle Aged , Multivariate Analysis , Neoplasm Recurrence, Local , Neoplasm Staging , PrognosisABSTRACT
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 (ΔYm) 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.
Subject(s)
Apoptosis , DNA-Binding Proteins/metabolism , Epithelial Cells/cytology , Epithelial Cells/metabolism , Homeostasis , Lens, Crystalline/cytology , Lysosomes/metabolism , Transcription Factors/metabolism , Animals , HEK293 Cells , Heat Shock Transcription Factors , Humans , Hydrogen-Ion Concentration , Lysosomes/chemistry , Mice , Mitochondria/metabolism , Up-Regulation , alpha-Crystallin B Chain/metabolismABSTRACT
Heat shock factor 4 (HSF4) is an important transcriptional factor that plays a vital role in lens development and differentiation, but the mechanism underlying the regulation of HSF4 is ambiguous. BCAS2 was reported to be an essential subunit of pre-mRNA splicing complex. Here, we identified BCAS2 as a novel HSF4 interacting partner. High expression of BCAS2 in the lens epithelium cells and the bow region of mouse lens was detected by immunohistochemistry. In human lens epithelial cells, BCAS2 negatively regulates HSF4 protein level and transcriptional activity, whereas in BCAS2 knockdown cells, HSF4 protein stability was increased significantly. We further demonstrated that the prolonged protein half-time of HSF4 in BCAS2 knockdown cells was due to reduced ubiquitination. Moreover, we have identified the lysine 206 of HSF4 as the key residue for ubiquitination. The HSF4-K206R mutant blocked the impact of BCAS2 on HSF4 protein stability. Taken together, we identified a pathway for HSF4 degradation through the ubiquitin-proteasome system, and a novel function for BCAS2 that may act as a negative regulatory factor for modulating HSF4 protein homeostasis.
Subject(s)
DNA-Binding Proteins/genetics , Epithelial Cells/metabolism , Lens, Crystalline/metabolism , Neoplasm Proteins/genetics , RNA Splicing , Transcription Factors/genetics , Ubiquitination , Animals , Cell Line , Complex Mixtures/chemistry , DNA-Binding Proteins/metabolism , Epithelial Cells/cytology , Heat Shock Transcription Factors , Humans , Lens, Crystalline/cytology , Mice , Mice, Inbred BALB C , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/metabolism , Proteasome Endopeptidase Complex/metabolism , Protein Binding , Protein Stability , Proteolysis , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Signal Transduction , Transcription Factors/metabolism , Transcription, GeneticABSTRACT
The eye and lens represent excellent models to understand embryonic development at cellular and molecular levels. Initial 3D formation of the eye depends on a reciprocal invagination of the lens placode/optic vesicle to form the eye primordium, i.e., the optic cup partially surrounding the lens vesicle. Subsequently, the anterior part of the lens vesicle gives rise to the lens epithelium, while the posterior cells of the lens vesicle differentiate into highly elongated lens fibers. Lens fiber differentiation involves cytoskeletal rearrangements, cellular elongation, accumulation of crystallin proteins, production of extracellular matrix for the lens capsule, and degradation of organelles. This chapter summarizes recent advances in lens development and provides insights into the regulatory mechanisms and differentiation at the level of chromatin structure and dynamics, the emerging field of noncoding RNAs, and novel strategies to fill the gaps in our understanding of lens development.
Subject(s)
Crystallins/genetics , Gene Expression Regulation, Developmental , Lens, Crystalline/embryology , Lens, Crystalline/metabolism , Animals , Cell Differentiation/genetics , Crystallins/metabolism , Gene Regulatory Networks , Humans , Models, BiologicalABSTRACT
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.
Subject(s)
DNA-Binding Proteins/physiology , Epithelial Cells/physiology , G1 Phase Cell Cycle Checkpoints/genetics , Lens, Crystalline , Transcription Factors/physiology , Tumor Suppressor Protein p53/metabolism , Cell Differentiation/genetics , Cell Proliferation/genetics , DNA-Binding Proteins/metabolism , Epithelial Cells/cytology , Genes, Switch , Heat Shock Transcription Factors , Humans , Lens, Crystalline/cytology , Lens, Crystalline/physiology , Mutant Proteins/metabolism , Protein Binding , Protein Stability , Transcription Factors/metabolism , Transcriptional Activation/genetics , Tumor Cells, CulturedABSTRACT
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.
Subject(s)
DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/physiology , Epithelial Cells/physiology , Lens, Crystalline/cytology , Transcription Factors/antagonists & inhibitors , Transcription Factors/physiology , Animals , Cell Survival/genetics , Cells, Cultured , Down-Regulation , HSP70 Heat-Shock Proteins/genetics , HSP70 Heat-Shock Proteins/metabolism , Heat Shock Transcription Factors , Heat-Shock Proteins/genetics , Heat-Shock Proteins/metabolism , Homeostasis/genetics , Lens, Crystalline/physiology , Mice , Molecular Chaperones , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Transcriptional ActivationABSTRACT
PURPOSE: The clinical management of cataracts in infancy involves surgical removal of the lens to ensure transmission of light to the retina, which is essential for normal neural development of the infant. This surgery, however, entails a lifelong follow-up and impaired vision. To our knowledge, no animal models recapitulate human lamellar opacities, the most prevalent form of early childhood cataracts. We present data on the recreation of the human lamellar cataract phenotype in transgenic mice. METHODS: Mutations in the DNA binding domain (DBD) of the heat shock transcription factor 4 (HSF4) are known to be associated with early childhood autosomal dominant lamellar cataract. We used bacterial artificial chromosome (BAC) transgenesis to express a hybrid gene: Hsf4 (DBD)-enhanced green fluorescent protein (EGFP), by recombineering EGFP sequences into the DBD of the Hsf4 gene, to interfere with the DNA binding properties of Hsf4. RESULTS: We recapitulated the human lamellar cataract, in its temporal as well as spatial presentation, within the transgenic mouse lens. This phenotype was reproduced faithfully using four different BACs, indicating that EGFP can be used to target transcription factor function in transgenic mice. Molecular and cell biological examination of early postnatal transgenic lens reveals impairment of secondary fiber cell differentiation. CONCLUSIONS: Recreation of the human lamellar cataract phenotype in mice allows investigation of this human pathology at a level not possible previously and points to the relevance of fiber cell heterogeneity dictated by fiber cell-specific gene activity in the biogenesis of the lamellar cataract.
Subject(s)
Cataract/genetics , DNA-Binding Proteins/genetics , DNA/genetics , Gene Expression Regulation, Developmental , Genes, erbB-1/genetics , Transcription Factors/genetics , Animals , Cataract/metabolism , Cataract/pathology , Cells, Cultured , Child , DNA-Binding Proteins/biosynthesis , Disease Models, Animal , Genotype , Heat Shock Transcription Factors , Humans , Immunoblotting , Mice , Mice, Transgenic , Real-Time Polymerase Chain Reaction , Transcription Factors/biosynthesisABSTRACT
Our previous study identified five new heat shock factor 4 (HSF4) mutations in 150 age-related cataract (ARC) patients which indicated that HSF4 mutations may be associated with this disease. Hypoxia-inducible factor (Hif1α) is an important downstream target of HSF4b. It has been found that Hif1α play also important roles in cataract development. To identify if HSF4b play it role in cataract development through HIF1α, we transfected SRA01/04 lens epithelial cells with small hairpin RNA of HSF4b and measured expressions Hif1α after transfection. Then, we perform chromatin immunoprecipitation quantitative PCR to see the relationship between HSF4b and HIF1α. We found that HSF4 downregulation led to decrease of HIF1α mRNA expression. Furthermore, we demonstrated by ChIP followed by quantitative PCR (ChIP-qPCR) that these HIF-1α is bound by HSF4b near promoters, not gene bodies.