title: Adenovirus-mediated overexpression of novel mutated IkappaBalpha inhibits nuclear factor kappaB activation in endothelial cells.
abstract: BACKGROUND
  Nuclear factor kappaB (NF-kappaB) overactivation, requiring phosphorylation and degradation of its inhibitor IkappaBalpha, is the basis for chronicity of airway inflammation in asthma. Based on our previous plasmid pShuttle-IkappaBalpha, carrying an IkappaBalpha gene from human placenta, we optimized a novel IkappaBalpha mutant (IkappaBalphaM) gene, constructed and characterized its replication-deficient recombinant adenovirus (AdIkappaBalphaM), and tested whether AdIkappaBalphaM-mediated overexpression of IkappaBalphaM could inhibit the NF-kappaB activation in endothelial cells.
  
  
  METHODS
  IkappaBalphaM gene (203 - 1003 bp) encoding 267 amino acids, acquired by site-directed deleting N-terminal phosphorylation sites of serine 32/36, was subcloned into the pShuttle and pGEM-T vectors for further polymerase chain reaction (PCR), restriction digestion, deoxyribonucleic acid (DNA) sequencing and homology analyses. Subsequent to inserting the expression unit of pShuttle-IkappaBalphaM, containing cytomegalovirus (CMV) promoter, IkappaBalphaM complementary DNA (cDNA) and polyadenylic acid (PolyA) signals, into the type 5 adenovirus (Ad5) vector, the resultant AdIkappaBalphaM was packaged in human embryonic kidney (HEK) 293 cells by cotransfection with lipofectamine. Western blot analysis and electrophoretic mobility shift assay were utilized to detect the AdIkappaBalphaM-mediated overexpression of IkappaBalphaM in HEK293 cells and its suppressive effect on phorbol 12-myristate 13-acetate (PMA)-induced NF-kappaB activation in human umbilical vein endothelial (ECV304) cells, respectively.
  
  
  RESULTS
  The relevant nucleotides and deduced amino acids of 801 bp IkappaBalphaM gene were consistent with those of IkappaBalpha gene (GenBank accession number: M69043). The titer of the prepared AdIkappaBalphaM was 4.0 x 10 (12) plaque-forming units (pfu)/L. Moreover, the IkappaBalphaM gene was overexpressed in HEK293 cells, and potently inhibited the PMA-induced NF-kappaB activation in ECV304 cells dose-dependently.
  
  
  CONCLUSIONS
  AdIkappaBalphaM is a novel vector for both efficient transfer and specific overexpression of IkappaBalphaM gene, as well as potent inhibition of NF-kappaB activity, providing a promising strategy for gene therapy of asthma.
authors.affiliation: Department of Respiratory Medicine, First Affiliated Hospital, Nanjing Medical University, Nanjing 210029, China.
authors.forename: Lin-fu
authors.initials: LF
authors.lastname: Zhou
authors.forename: Kai-sheng
authors.initials: KS
authors.lastname: Yin
authors.forename: Zi-lu
authors.initials: ZL
authors.lastname: Zhu
authors.forename: Yi
authors.initials: Y
authors.lastname: Zhu
authors.forename: Xin
authors.initials: X
authors.lastname: Yao
authors.forename: Hui
authors.initials: H
authors.lastname: Mao
authors.forename: Wei-ping
authors.initials: WP
authors.lastname: Xie
authors.forename: Mao
authors.initials: M
authors.lastname: Huang
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D002460:Cell Line; D042783:Endothelial Cells / Q000378:metabolism*; D015316:Genetic Therapy; D006801:Humans; D020672:I-kappa B Proteins / Q000235:genetics*; D009154:Mutation; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D000072000:NF-KappaB Inhibitor alpha; D013755:Tetradecanoylphorbol Acetate