The Hsf4b Transcription Activity Is Regulated By Phosphorylation Of Serine15

BackgroundThe incidence of congenital cataract, the main cause of blindness in children, is approximately0.3-1.5per1000children in the world. Histopathological alterations of congenital cataract features retarded lensdevelopment, non-clear scar formation in lens. Chromosomal abnormalities are the main causes ofcongenital cataracts[1]. A large amount of data show that mutations and functional defects of transcriptionfactor gene are one of hereditary causes, such as Pax6, FoxE3, Six3, Prox1, Sox2/3, Maf, Pitx3, AP-2a andHsf4[2-4].Under normal circumstances, the instantaneous activation and inactivation of these transcriptionfactors determines the different developmental stages of lens, e.g. activation of Pax6in cells on the surfaceof neuronal ectodem at early stage of embryo promote ectodermal epithelial cells to form the lens placodeand architecture of lens vesicle, induce epithelial cell differentiation into fiber cells, and control proteinexpression of delta-crystallin, alpha A-crystallin, etc[5,6]. Pax6transcriptional activity is suppressed afterbirth with the maturity of the lens. Pax6gene mutations are closely related with clinical congenitalcataract[7]. Heat shock transcription factor4(Hsf4) is one of key transcription factors that regulate the lensdevelopment during neonatal period. Under normal circumstances, Hsf4protein expression starts at E13.5of embryo, reaches maximum during neonatal period (P1-P28) and gradually declines as the lensmature[8-10]. Mis-sense mutations of Hsf4DNA binding domain (e.g. L115P, I87V, A20D, and H74R) isclosely correlated with familial autosomal dominant hereditary lamellar cataract in China and Denmark[4],and familial dominant hereditary whole cataract (whole whitemu cataract)[11]. The Hsf4knockout micecould induce congenital cataracts in neonatal mice with the main pathological changes including abnormalproliferation of anterior lens capsule epithelium, accumulation of denatured proteins in fibroblast cells andterminal differentiation defects of fiber cells[8-10]. It is generally regarded that Hsf4transcriptional activityis essential to maintain the normal development of lens during neonatal period.HSF4, one of the members of the of heat shock transcription factors family, includes two isotypes,Hsf4a and Hsf4b, in organism, which were derived from the different splicing of the same gene. Due to different structures, HSF4a and Hsf4b have distinct transcriptional activity, i.e. transcriptional suppressionfor HSF4a and transcriptional activation for Hsf4b, respectively. Hsf4b is the only active form in lens. It ismainly involved in expression regulation of small heat shock proteins (Hsp25, r-crstallin, a-crystallin) andcytoskeletal proteins (imentin, filesin). The transcriptional activity of Hsf4is regulated by Hsf4phosphorylation but the mechanisms of Hsf4phosphorylation regulation and related signaling pathways arenot understood. Understanding of Hsf4b phosphorylation transcription regulation is important to reveal thecorrelation of Hsf4dysfunction and congenital cataract. By using glycine site-directed mutagenesistechniques, the potential phosphorylation sites of Hsf4b were scanned and found that mutation in Hsf4bDNA binding domain at15serine residual has remarkable inhibitory effect on Hsf4b transcription activity.The present study explore the Hsf4b transcriptional activity regulation by S15phosphorylation byestablishment of Hsf4b15loci mutation expression vector and subsequent stable expression cell lines oflens epithelial cells from mice.ObjectivesTo investigate the S15phosphorylation on transcriptional activity of Hsf4b regulation.Methods1. According to the published Hsf4b gene sequence of mice in Gene bank, the software of premier primer5and GPS2.1phosphorylation site detection were used for mutated Hsf4b/S15. The primers used in thepresent study were as follows,Primer S15upstream primer: GAG CCA GGC CCC GCC CCC GTG CCT GCC;Primer S15downstream primer: GGC AGG CAC GGG GGC GGG GCC TGG CTC.PCR amplification was used for Hsf4b DNA binding domain cloning and inserted intopWZL-HA-Hsf4b-S15A followed by sequencing to confirm authenticity.2. Mouse lens epithelial cells mLEC/hsf4-/-was infected with retrovirus carrying wild Hsf4b and mutatedHsf4b/S15A, respectively, followed by blasticidin exposure for stable subline selection.3. Western blotting was used to verify the effects of downstream protein expression after4. Luciferase experiment was used to determine the effects of Hsf4bå’ŒHsf4b/S15A mutants on regulationof alpha B-crystallin gene promoter.Results 1. pWZL-HA-Hsf4b-S15A was cloned successfully and stable cell lines of mouse lens epithelial cells(MLEC) with stable expressin of pWZL-HA-Hsf4b-S15A was established.2. Compared with wild-type Hsf4b, mutated Hsf4b-S15A can significantly inhibit the expression ofdownstream protein HSP25, alpha B-crystallin and alpha A-crystallin.3. Mutated Hsf4b at15residule can inhibit transcriptional activity of the alpha B-crystallin gene promoter.ConclusionHsf4b S15phosphorylation is important for the transcriptional regulation of Hsf4b. S15AHsf4bmutatants can significantly inhibit the protein expression of downstream protein HSP25, alpha B-crystallinand alpha A-crystallin. Hsf4b S15Phosphorylation is involved in regulation of downstream gene promoter.