RESUMO
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.
Assuntos
Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Treonina/genética , Treonina/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Linhagem Celular , Núcleo Celular/genética , Expressão Gênica , Células HEK293 , Proteínas de Choque Térmico HSC70/genética , Proteínas de Choque Térmico HSC70/metabolismo , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico HSP27/metabolismo , Fatores de Transcrição de Choque Térmico , Proteínas de Choque Térmico , Humanos , Chaperonas Moleculares , Mutação/genética , Fosforilação , Transporte Proteico , Transcrição Gênica , Cadeia B de alfa-Cristalina/genética , Cadeia B de alfa-Cristalina/metabolismo , beta Carioferinas/genética , beta Carioferinas/metabolismoRESUMO
OBJECTIVE: To establish mouse lens epithelial cell lines with the genotype of Hsf4-/-. METHODS: The expended mouse lens epithelial cells, which were generated from P6 Hsf4-deficient mouse lens epithelia, were immortalized with SV40-T-antigen and named MLEC/Hsf4-/- cell. The expression of alpha A-crystallin was immunoblotted. Hsf4b cDNA was reconstituted by transiently transfection. RESULTS: The SV40-immortalized cells were in adherent growth mode with spindle morphology, pseudopodia, clear nuclear boundary membrane and cytoplasm translucent. Immunoblotting results indicated that the lens biomarker protein alpha A-crystallin was expressed in MLEC/Hsf4-/- cells. Reconstitution of Hsf4b into MLEC/Hsf4-/- cells upregulated the expression of Hsp25. CONCLUSIONS: The SV40-immortalized MLEC/Hsf4-/- cells have the lens epithelial characteristics and could be used as a tool for studying the signal transduction in vitro.
Assuntos
Linhagem Celular , Células Epiteliais/citologia , Cristalino/citologia , Animais , Proteínas de Ligação a DNA/genética , Fatores de Transcrição de Choque Térmico , Camundongos , Fatores de Transcrição/genéticaRESUMO
AIM: To investigate the genetic mutations that are associated the hereditary autosomal dominant cataract in a Chinese family. METHODS: A Chinese family consisting of 20 cataract patients (including 9 male and 11 female) and 2 unaffected individuals from 5 generations were diagnosed to be a typical autosomal dominant cataract pedigree. Genomic DNA samples were extracted from the peripheral blood cells of the participants in this pedigree. Exon sequence was used for genetic mutation screening. In silico analysis was used to study the structure characteristics of connexin 46 (CX46) mutant. Immunoblotting was conduceted for testing the expression of CX46. RESULTS: To determine the involved genetic mutations, 11 well-known cataract-associated genes (cryaa, cryab, crybb1, crybb2, crygc, crygd, Gja3, Gja8, Hsf4, Mip and Pitx3) were chosen for genetic mutation test by using exon sequencing. A novel cytosine insertion at position 1195 of CX46 cDNA (c.1194_1195ins C) was found in the samples of 5 tested cataract patients but not in the unaffected 2 individuals nor in normal controls, which resulted in 30 amino acids more extension in CX46C-terminus (cx46fs400) compared with the wild-type CX46. In silico protein structure analysis indicated that the mutant showed distinctive hydrophobicity and protein secondary structure compared with the wild-type CX46. The immunoblot results revealed that CX46 protein, which expressed in the aging cataract lens tissues, was absence in the proband lens. In contrast, CX50, alpha A-crystallin and alphaB-crystallin expressed equally in both proband and aging cataract tissues. Those results revealed that the cx46fs400 mutation could impair CX46 protein expression. CONCLUSION: The insertion of cytosine at position 1195 of CX46 cDNA is a novel mutation site that is associated with the autosomal dominant cataracts in this Chinese family. The C-terminal frameshift mutation is involved in regulating CX46 protein expression.
RESUMO
Heat shock factor 4b has been found to be closely associated with postnatal lens development. It expresses in postnatal lens epithelial and secondary fiber cells and controls the expression of small heat shock proteins which are important for lens homeostasis. However, the signal pathways underlying Hsf4b are still not completely understood. Here we present that Hsf4b transcription activity is regulated by FGF2 a key growth factor that is involved in regulating lens development at multiple stages. FGF2 can promote Hsf4b nuclear-translocation and the expression of Hsp25 and αB-crystallin, the key downstream targets of Hsf4b in the Hsf4b-reconstituted mouse hsf4-/- lens epithelial cells. Further study indicates that FGF2 can induce Hsf4b protein stabilization through ERK1/2-mediated posttranslational phosphorylation or sumoylation. Hsf4b can promote FGF2-induced morphology transition from lens epithelial cell to the fiber cell, and this morphology transition can be inhibited by ERK1/2 inhibitor U0126. Taken together, our data demonstrate that Hsf4b is a novel downstream transcription factor of FGF2, and its transcription activity is associated with FGF2-modulated lens epithelial cell-fiber cell transition.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Fator 2 de Crescimento de Fibroblastos/fisiologia , Fatores de Transcrição/metabolismo , Transcrição Gênica , Ativação Transcricional , Animais , Núcleo Celular/metabolismo , Forma Celular , Células Epiteliais/fisiologia , MAP Quinases Reguladas por Sinal Extracelular , Células HEK293 , Fatores de Transcrição de Choque Térmico , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Cristalino/citologia , Cristalino/metabolismo , Camundongos , Camundongos Knockout , Chaperonas Moleculares , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Transporte Proteico , Proteólise , Sumoilação , Regulação para Cima , Cadeia B de alfa-Cristalina/genética , Cadeia B de alfa-Cristalina/metabolismoRESUMO
Heat shock factor 4 (Hsf4b) has been identified as a novel cataractogenic protein whose mutation has been closely associated with hereditary cataracts in humans and animals. It acts both as a transcription activator and a transcription inhibitor in the regulation of its downstream targets during lens development. However, the signaling factors that regulate Hsf4b transcription activity are still not completely defined. Here, we found that Hsf4b, not Hsf4a (another isoform of Hsf4), acts as the inhibitor of CMV promoter as well as the activator of Hsp25 in the Hsf4-/- mouse lens epithelial cell line (mLEC/hsf4-/-). Hsf4b inhibits CMV-promoter activity by directly binding to TTCC (HSE motif) at 173-176bps in the CMV promoter. The phosphorylation of Hsf4b/S299 in the PDSM motif, which is absent in Hsf4a, participates in the negative regulation of the CMV promoter. The transcriptional inhibition of Hsf4b is associated with transcriptional inhibitor Daxx. Hsf4b can interact and co-localize with Daxx in the nucleus, and their association is regulated by the phosphorylation of Hsf4b/S299. In addition, we found that Hsf4a and Hsf1 were also associated with Daxx. However, in contrast to activating Hsf1, Daxx can repress Hsf4b-induced expression of Hsp25 in the mLEC/hsf4-/- cell line. Our results demonstrate that the transcription-inhibitory function of Hsf4b is regulated by the phosphorylation of Hsf4b/S299 and phosphorylation-dependent association with Daxx.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Catarata/genética , Citomegalovirus/genética , Proteínas de Ligação a DNA/metabolismo , Células Epiteliais/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular Transformada , Proteínas Correpressoras , Proteínas de Ligação a DNA/genética , Regulação para Baixo/genética , Células Epiteliais/patologia , Técnicas de Silenciamento de Genes , Células HEK293 , Fatores de Transcrição de Choque Térmico , Humanos , Cristalino/patologia , Camundongos , Chaperonas Moleculares , Mutação/genética , Fosforilação , Regiões Promotoras Genéticas/genética , Ligação Proteica/genética , Fatores de Transcrição/genéticaRESUMO
AIM: Ton construct eukaryotic plasmid expressing Hsf4B and to investigate Hsf4b is phosphorylated by MAP kinase P38: METHODS: The total RNA of human heart tissues were prepared. Hsf4b cDNA were then synthesized with RT-PCR. The PCR products were digested with Kpn I and EcoR I and subcloned into pcDNA3.0, pcDNA-Flag-Hsf4b was transfected into HEK293T cells. The expression of Hsf4b was testified with Western blotting. The interaction between Hsf4b and P38 was assayed by immunoprecipitation. In vivo pull down GST demonstrated that Hsf4B (196-493) could interact with P38, P38 phosphorylation of Hsf4b were testified with Kinase assay. RESULTS: We subcloned the human cDNA of Hsf4b into eukaryotic expression vectors pcDNA3 and PEBG, and Hsf4b was overexpressed in HEK293T cells. Further studies demonstrated that Hsf4b could interact with and phosphorylated by MAP kinase P38. CONCLUSION: Hsf4b could interact with and phosphorylated by MAP kinase P38. Our results will provide more evidence for understanding the signal regulation of Hsf4b transcription activity during lens development.