| BackgroundCongenital cataract is one of main cause for children blindness.In the early stages of embryonic development,dysregulation of the genes,which results in abnormal development of the lens,cause 0.3-1.5/1000 of congenital cataracts in children.more than 30 causative genes which may lead to congenital cataract.Transcription Factors Gene mutations and functional defects which are one of the genetic causes of congenital cataracts such as Pax6,Fox E3,Six3,Prox1,Sox2 / 3,Maf,Pitx3,AP-2a and Hsf4.The activation and inactivation of these transcription factors determines the process of lens development.Hsf4 knockout mice and zebrafish can cause abnormal lens development and cataract-related phenotypes.The main pathological changes are an abnormal proliferation of the anterior lens capsule,degeneration of fibrin intracellular proteins,and terminal differentiation disorder of fibroblasts.Therefore,the transcriptional regul-ation of Hsf4 is essential to maintain the normal development of the neonatal lens,however,The heat shock factor4,which our regulatory mechanism of the transcriptional activity is unclear.The heat shock factor 4,which belongs to the heat shock factor family,has been found to be the fundamental transcription factor in the lens development at postnatal age.the family members,Which consists of Hsf1,Hsf2,Hsf3 and Hsf4 in mammalian tissues.Hsf4 has two isoforms,Hsf4 a and Hsf4 b.Hsf4 has two isoforms,Hsf4 a and Hsf4 b.Hsf4b is the unique isoform of Hsf4 that specifically expresses in mouse lens tissue.During lens development,It acts as a transcription activator,and positively controls the expression of Hsp25,and γ-crystallin,β-crystallin and structural protein vemintin,filensin and SKAP2.On the other hand,it also acts an inhibitor and negatively regulates the expression of FGF subfamily members.Our preliminary data demonstrate that Hsf4 b transcriptional activity is regulated by post-translational phosphorylation during lens development.The results of alanine-mutation scanning demonstrated that the phosphorylation of Hsf4b/T222 and S299 shows different regulatory effects on Hsf4 b transcription activity.Previous studies have shown that phosphorylation of Hsf4 b does not specifically phosphorylate antibodies to detect the phosphorylation level of Hsf4 b,and that phosphorylation of these protein kinases is not known at these sites.Studying the upstream kinase of Hsf4 b has important implications for establishing a signaling pathway that regulates Hsf4 b transcriptional activity and how Hsf4 b plays a role in the lens.The subject is based on the customization of phosphorylated antibodies at the Hsf4b/S299 locus,and discusses in depth the upstream kinases and signaling pathways of phosphorylation-modified Hsf4b/S299,as well as the molecular mechanisms by which they phosphorylate S299 to regulate the transcriptional activity of Hsf4 b.Based on this,we can provide new insights for us to deeply understand the transcriptional activity of phosphorylated Hsf4 b and the cataract occurrence.PurposeUsing the lens epithelial cells and lens of BL/C57 mice,we explore what kinase is involved in modifying the phosphorylation of the Hsf4b/S299 A site and the regulatory mechanism of Hsf4 b transcriptional activity,and its role in lens development.Methods 1.Preparation of p-Hsf4b/S299 antibody verified that lens epithelial cells overexpress wild-type Hsf4 protein with different tags.lens epithelial cells overexpress wild-type Hsf4 b protein with different tags by immunoprecipitation.lens epithelial cells over-express wild-type Hsf4 b and Hsf4 b /S299 A,Hsf4b/S298 D,and Hsf4b/T222 A mutant proteins;cells overexpressing wild-type Hsf4 b,we treat cell lysate supernatants with Calflate alkaline phosphatase(CIAP)the enzyme.All the above were the specific encognition and binding activity of phosphorylation of Hsf4b/S299 by Western-blot and phosphorylation of Hsf4b/S299 by phosphorylated Hsf4b/S299 antibody.2.Lens epithelial cells over-express the wild-type Hsf4 b and Hsf4b/S299 A mutations and treat the cells with cis respectively.Lens epithelial cells over-express the wild-type Hsf4 b and Hsf4b/S299 A mutations and treat the cells with MG132 respectively.Lens epithelial cells over-express wild-type Hsf4 b protein while treating cells with EGF.We detect phosphorylation of Hsf4b/S299 by Western-blot above all.3.we treat cells with EGF and then with U0126.We detect the best effect of U0126 by Western-blot.the first we treat the lens epithelial cells that overexpress wild-type Hsf4 b with EGF,and then with U0126.Lens epithelial cells over-express the wild-type Hsf4 b and Hsf4b/S299 A mutations and treat the cells with cis respectively.Cells that overexpress wild-type Hsf4 b treat with U0126,SB203580, and SP600125 respectively.We treat the lens epithelial cells that over-expressed the wild-type Hsf4 b and Hsf4b/S299 A mutant with MG132,while treating that overexpress wild-type Hsf4 b cells with U0126.We detect phosphorylation of Hsf4b/S299 by Western-blot above all.4.Lens epithelial cells overexpress wild-type Hsf4b(WT),mutate Hsf4b/S299 A and Hsf4b+MEK1 protein,we treat Hsf4b+MEK1 cells with U0126,then detect the phosphorylation levels of Hsf4b/S299 and ERK1/2 by Western-blot.5.HLE(human lens epithelial)cells overexpress wild-type Hsf4 b,Hsf4b+MEK1,Hsf4b/S299 A mutations,and Hsf4b/S299 A mutations+MEK1 protein;HLE cells overexpress Sumo1+ Hsf4 b,Sumo1+ Hsf4b/S298 A,Sumo1+ Hsf4b/ S298A/K294 R,Sumo1+ Hsf4b+MEK1 protein,we treat overexpress Sumo1+Hsf4b+MEK1 cells with U0126,detect phosphorylation of Hsf4b/S299 and ERK,and the expression of downstream target gene alpha B-crystallin and Hsf4b/ K294 ubiquitination by Western-blot.6.We harvest the lenses of C57 mice for 3 days,7 days,2 weeks,and 2 months,then detect the phosphorylation levels of Hsf4 b and Hsf4b/S299 proteins at different time point by Western-Blot.We remove one week old mouse lens which epitheliμm and fibers were separated,then perform the nucleoplasm isolation assay and detect the protein expression of Hsf4 b and p-Hsf4b/S299 in the nucleus and cytoplasm of epithelial and fibroblast cells by Western-blot,respectively.Two-week-old mouse lenses removed and sectioned,he localization of phosphorylated Hsf4/S299 protein in epithelial and fibroblasts was detected by immunofluorescence.7.We treat the lens of one-week-old mice with EGF and U0126.then detect Hsf4 b,p-Hsf4b/S299,and its downstream target proteins by Western-blot.Results 1.The prepared Hsf4b/S299 phosphorylated antibodies specifically encognize phosphorylated proteins of differently tagged Hsf4b/S299 sites.2.U0126 can inhibit the phosphorylation of Hsf4b/S299 sites induced by cisplatin,MG132 and EGF.3.MEK1 can mediate activation of ERK1/2,which in turn causes phosphorylation of the Hsf4b/S299 locus.4.The MEK1-ERK1/2 pathway can increase the phosphorylation of the Hsf4b/S299 site,decrease the expression of its downstream target gene alpha B-crystallin,and activate the ubiquitination of the Hsf4b/K294 locus.5.Spatiotemporal expression of Hsf4 b and p-Hsf4b/S299 in the lens of C57 mice.p-Hsf4b/S299 is mainly expressed in the cytoplasm of mouse lens epithelial cells and the nuclei of fibrocytes.6.In mouse lens tissue,MAP kinase ERK1/2 is also involved in the phosphorylation of Hsf4b/S299,which in turn regulates the transcriptional activity of Hsf4 b.Conclusions 1.In lens epithelial cells,MEK1 mediates phosphorylation of ERK1/2,thereby increasing the phosphorylation level of Hsf4b/S299.2.Phosphorylation of the Hsf4b/S299 locus occurs through activation of the MEK1-ERK1/2 signaling pathway,which in turn causes the expression of its downstream target gene alpha B-crystallin to decline.3.During the development of the lens,phosphorylation of the Hsf4b/S299 locus can regulate the transcriptional activity of Hsf4 b. |
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