Regulatory Effect Of Hsf4b/S299 Phosphorylation On Its Transcriptional Activity

Background The arbitrary degree of opacities in lens of the eye is named cataract.Abnormal change of the cellular structure of lens epithelial or fibroblast cell can lead to the occurrence of cataract in the processes of lens development. Cataract is a major cause of human blindness. According to its occuring ages, cataract can be divided into congenital cataract and age-related cataract. It is reported that 60-70% of people aging over 50 suffer from cataract. Therefore, with aging people expanding, therapeutic prevention of age-related cataract has become urgent scientific problem. In response to the variety of intracellular and extracellular stresses, the cell can protect itself from the damage by upregulating the expression of heat shock protein(Hsps) expression, which work as molecular chaperones participating in remodeling the protein refolding, distribution and degradation. The expression of heat shock proteins in mammalian cells is regulated by a small family of heat shock factors( Hsf1, Hsf2, Hsf3 and Hsf4). Heat shock transcription factor 4( HSF4) is a member of heat shock factor family(HSF).Hsf4 has two splicing variants: Hsf4 a and Hsf4 b.Genetic analysis showed that the mutations in Hsf4 is associated with the incidence of congenital cataract and age-related cataract. Hsf4 knockout in mice could lead to neonatal congenital cataract., characterizing with The mice with Hsf4 knocked out, lead to the abnormal proliferation of lens epithelial cell and fibroblast differentiation. These results indicate that Hsf4 is involved in the regulation of lens fibroblast differentiation in mouse.The study found that Hsf4 is highly express in lens cells, and Hsf4 b is the unique form of Hsf4 that specifically expresses in mouse lens tissue. Hsf4 b mainly exists in lens epithelial cells and secondary fiber cells and plays an important role during the formation and differentiation of lens. Our previous study showed that the Hsf4 b protein can be regulated by phosphorylation and, sumoylation.. Phosphorylation of Hsf4b/T472 is involved in the regulation of Hsf4 b translocation from cytoplasm to nucleus by assisting association of Hsf4 b with nuclear transporter factor inportin beta-1..Our previous study also showed that Hsf4b/S299 phosphorylation regulates the interaction between Hsf4 b and transcriptional inhibitor DAXX. Hsf4b/S299 phosphorylation can repress the transcriptional activity of Hsf4 b. Lea Sistonen found that Hsf4 b can also be sumoylated to regulate transcriptional activity of Hsf4 b.The sumoylation of Hsf4 b depends on the phosphorylation of Hsf4b/S299, and in other words, there is a phosphorylation dependent sumolation motif, PDSM of Hsf4 b. Since the conclusions of inhibitory role of phosphorylation of Hsf4b/S299 are come from the gal-reporter system in the tumor cell line, it is not know how the phosphorylation of Hsf4b/S299 participates in regulating it natural downstream targets in lens epithelial cells. Our previous results indicate that Hsf4 b can upregulate the expression of Hsp25 and alpha B-crystallin and simultinously inhibit the expression of Hsp70 in mouse lens epithelial cells. In this study, we established the Hsf4b/S299A(D) mutations by using site-mutation methods, and make lens epithelial cell stable cell lines that express Hsf4b\Hsf4b/S299A、Hsf4b/S299 D. The regulatory effect of S299A(D) on Hsf4 b are studied.Objectives To determine how the phosphorylation of S299 regulates Hsf4 b transcription activity in lens epithelial cell line.Methods 1.Hsf4b/S299 mutant primers are designed; we constructed Hsf4b/S299 A and Hsf4b/S299 D mutations of p WZL-blast-HA-Hsf4b/S299 A and p WZL-blast-HA-Hsf4b/S299 D by site directed mutagenesis method. 2.After mutation plasmids were successful constructed, the Hsf4b/S299 mutants plasmids and normal plasmid p WZL-blast-HA-Hsf4 b were transfected into the retoviral packaging cell line 293 phoenix cells respectively to making the recombinant retrovirus.The supernatants of 293 phoenix cells, which contain the virus, were applied to infect the m LEC/Hsf4b-/- cells. Finally, we generated the stable cell lines by using drugs(Blasticidin) selection and obtain sustainable wild type and mutant Hsf4 b expression of the stable cell line. 3.Western blot and realtime-PCR assays detect the influence of the mutantions of Hsf4b/S299 on the expression of downstream protein(hsp25、αB-crystallin and Hsp70). 4.The wild-type Hsf4 b and mutation Hsf4b/S299 stable cell lines were treated with CHX to determine whether the Hs4b/S299 mutants have influence on Hsf4 protein stability. 5.To determine their regulatory effect on Hsp70 and alha B-crystallin promoters by using the Luciferase assay.6.CHIP assay detects the association of the Hsf4 b 、 Hsf4b/S299 A and Hsf4b/ S299 D with downstream targets promoter. 7.Cell immunofluorescence assay detects the influence of Hsf4 b cellular localization in Hsf4b/ S299 A and Hsf4b/S299 D stable cell line.Results 1.The mutation plasmids of p WZL-blast-HA-Hsf4b/S299 A 、 p WZL-blast-HA-Hsf4b/S299 D 、p WZL-blast-HA-Hsf4b/K294 R were successful constructed. 2.The stable strains of lens epithelial cell lines that could express sustained and stable wild-type Hsf4 b and mutant Hsf4b/S299A、Hsf4b/ S299 D were successful established. 3.Experiments of realtime-PCR show that Hsf4 b could upregulate the m RNA leval of small heat shock protein hsp25 and αB-crystallin, the mutantions of Hsf4b/S299 A further increases the ability to upregulate the m RNA leval of hsp25 and αB-crystallin. In contrary, mutantions of Hsf4b/S299 A and Hsf4b/S299 D lose the ability to downregulate the expression of Hsp70. 4. Experiments of Western blot show that Hsf4 b could upregulate the protein leval of small heat shock protein hsp25 and αB-crystallin, Meanwhile the mutantions of Hsf4b/S299 A could significantly increase their protein levels further, on the contrary, Hsf4b/S299 D cannot. At the same time, Hsf4 b could downregulate protein levels of Hsp70, but Hsf4b/S299 A lose the ability to downregulate the protein level of Hsp70, Hsf4b/S299 D has no obvious influences on the expression of Hsp70. 5.Compared with the wild-type Hsf4 b and mutant Hsf4b/ S299 A, and Hsf4b/S299 D, Hsf4b/S299 A protein stability is reduced, Hsf4b/S299 D protein compared with Hsf4b/S299 A protein is stable. 6.Experiments of luciferase assay and CHIP assay show that the mutantions of Hsf4b/S299 A and Hsf4b/S299 D could affect its DNA binding ability of Hsf4 b with its gene promoter of hsp70 and αBcrystallin.Hsf4b/S299 A mutantion enhanced its DNA binding ability, Hsf4b/S299 D mutantion has no effect on its DNA binding ability. 7.The cellular localization of Hsf4 b is not changed in lens epithelial stable strains of Hsf4b/S299 A and Hsf4b/S299 D mutantion.Conclusions In the lens epithelial cells, Hsf4 b can increase the expression of Hsp27 and a B-crystallin while suppressing the expression of Hsp70. Phosphorylation of Hsf4b/S299 can regulate expression levels of Hsp25 and a B-crystallin, while relieving inhibition of Hsf4 b regulation of Hsp70. Phosphorylation of Hsf4b/S299 exercises these functions through stabilization Hsf4 b protein and its DNA binding ability.