NFκB Signaling Activation Increases Human USP16 Gene Transcription

Objective Ubiquitin Specific Peptidase 16(USP16)plays many roles in gene expression,cell cycle progression,cell self-renewal and/or senescence pathways,its overexpression is a major facilitator of neurological deficits observed in Down’s syndrome,and it is closely related to neural stem cell defects,but the transcriptional regulation of USP16 gene is largely unknown.In this study,we focused on the transcriptional regulation of human USP16 gene by the transcription factor NFκB and its molecular mechanism.Materials and methods Primers design,plasmids construction,cell culture and transfection,dual-luciferase assay,nucleoprotein and Ch IP DNA extraction,electrophoretic mobility shift assay,Chromatin immunoprecipitation assay,cell intervention,quantitative Real-time PCR.Results 1.The 2,324 bp 5’ flanking region of the USP16 gene was cloned into the promoter-lacking plasmid vector p GL4.10 to generate p USP16-A(-1856 to +468),and a series of deletion within p USP16-A was generated.The luciferase assays of these deletion plasmids were performed,a series of positive and negative regulatory regions were obtained.2.The human USP16 gene promoter was shown to contain several putative regulatory elements,such as NFkB,HIF,and SP1 responsive elements.3.Four human USP16 promoter deletion constructs,p USP16-N1~N4,were cloned into p GL4.10 vector.Compared with empty vector,NFkB p65 expression plasmid co-transfected with p USP16-N1,p USP16-N2,p USP16-N3 and p USP16-N4,resulted in enhanced luciferase activities to about 2 fold,2.69,2.51,2.24 and 2.04 fold,respectively(p<0.001),and the statistical significance was achieved in N1 vs.N3,N1 vs.N4 and N2 vs.N4,these suggests deletion of binding site 2 and 3 have huge impact on NFkB’s role in upregulating USP16’s gene promoter.In SH-SY5 Y cells,similar results were observed(p<0.001).4.By using EMSA,we found that predicted binding sites 2,3,4 bind physically with NFkB p65.Ch IP assay clearly demonstrated that predicted binding site2 and 3 can bind with NFkB p65,while site 1 and 4 can not.6.Stimulation of LPS resulted in a marked increase of endogenous USP16 m RNA levels in SH-SY5 Y cells(1.58-fold,p<0.05),but no significant effect in HEK293 cells(data not shown).However,TNFα enhanced the levels of endogenous USP16 m RNA both in HEK293 cells(1.90-fold,p<0.01)and SH-SY5 Y cells(1.85-fold,p<0.05).Conclusion The 5’ flanking region of USP16 gene,from-1856 to +468,showed promotor activity.NFkB can up-regulate the human USP16gene promotor activities.Only two binding sites(-826~-815 and-510~-501)interacts with p65.Activation of NFkB signaling by p65 overexpression,LPS and TNFα stimulus increases USP16 transcription.In conclusion,NFkB increases USP16 gene transcription through two cis-acting elements.Objective To analyze the characteristics of surfactant-associated protein A(SP-A)gene sequence and its bioinformatics characteristics and to investigate the m RNA and protein level of SP-A in lung injury model of cotton rats,so as to explore the correlated between lung injury and SP-A expression.Materials and methods A total of 32 cotton rats were randomly divided into control(normal saline,NS)group and three lipopolysaccharide(LPS)-induced lung injury groups(the cotton rats were injected intraperitoneally with 2mg/kg LPS for 24,48 and 96 hours,respectively).The total RNA was extracted from lung tissue and SP-A gene was amplified by RT-PCR.Then bioinformatics analysis was done for SP-A characteristics.Histopathological changes of lung tissue were observed at different time point by hematoxylin-eosin staining;the m RNA level of SP-A was detected by real time-PCR and the protein level of SP-A was detected by Western blotting analysis.Results The coding region of Cotton rat SP-A gene had 744 bp and encoded 248 amino acids,containing six cysteine residues,four α-helices and two predicted N-glycosylation sites.Meanwhile,cotton rat SP-A shared high level of homology in the nucleotide sequences(75.4%~90.1%)and in deduced amino acid sequences(70.6%~87.1%)with other species.Moreover,histopathological changes of lung tissue in three LPS groups were more notable and severe than those in the control group,and the changes were related to LPS treatment time.Compared with the control group,the m RNA and protein levels of SP-A in lung tissues were significantly increased in three LPS groups,with rapid increase starting from 24 h after treatment(p<0.001 and p<0.01),with continuous increase at 48h(p<0.001 and p<0.01),and slight decrease and still keeping at a high level at 96h(P<0.05 and P<0.01)Conclusion SP-A gene of cotton rat is highly conservative;the m RNA and protein levels of SP-A are closely associated with the severity of lung injury.