title: Cyclophilin A (CypA) interacts with NF-κB subunit, p65/RelA, and contributes to NF-κB activation signaling.
abstract: BACKGROUND
  Peptidyl-prolyl isomerase cyclophilin A (CypA) plays important roles in signaling, protein translocation, inflammation, and cancer formation. However, little is known about the mechanisms by which CypA exerts its effects. C57BL/6 Ppia (encoding CypA)-deficient embryonic fibroblasts show reduced activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), the p65/RelA subunit, suggesting that CypA may mediate modulation of NF-κB activity to exert its biological effects.
  
  
  METHODS
  Western blotting and qRT-PCR analyses were used to evaluate the association of CypA deficiency with reduced activation of NF-κB/p65 at the protein level. GST pull-down and co-immunoprecipitation were used to examine interactions between CypA and p65/RelA. Truncation mutants and site-directed mutagenesis were used to determine the sequences of p65/RelA required for interactions with CypA. Enhancement of p65/RelA nuclear translocation by CypA was assessed by co-transfection and immunofluorescent imaging. Treatment of cells with cycloheximide that were harvested at various time points for Western blot analyses was carried out to evaluate p65/RelA protein stability. The functional activity of NF-κB was assessed by electrophoretic mobility-shift assays (EMSA), luciferase assays, and changes in expression levels of target genes.
  
  
  RESULTS
  GST pull-down assays in vitro and co-immunoprecipitation analyses in vivo provided evidence for protein-protein interactions. These interactions were further supported by identification of a CypA-binding consensus-like sequence within NF-κB subunit p65 at the N-terminal 170-176 amino acid residues. Significantly, CypA provided stability for NF-κB p65 and promoted NF-κB p65 nuclear translocation, resulting in increased nuclear accumulation and enhanced NF-κB activity.
  
  
  CONCLUSIONS
  Our findings revealed important mechanisms that regulate NF-κB activation, and offer new insights into the role of CypA in aberrant activation of NF-κB-mediated signaling for altered expression of its target genes, resulting in pathological effects in various diseases.
authors.affiliation: Dental and Craniofacial Research Institute and School of Dentistry, University of California, Los Angeles, CA, United States of America.
authors.forename: Shan
authors.initials: S
authors.lastname: Sun
authors.affiliation: Dental and Craniofacial Research Institute and School of Dentistry, University of California, Los Angeles, CA, United States of America; Department of Neurosurgery, the 2nd Affiliated Hospital of Harbin Medical University, Harbin, Heilonjiang, China.
authors.forename: Mian
authors.initials: M
authors.lastname: Guo
authors.affiliation: Dental and Craniofacial Research Institute and School of Dentistry, University of California, Los Angeles, CA, United States of America.
authors.forename: James Beiji
authors.initials: JB
authors.lastname: Zhang
authors.affiliation: Dental and Craniofacial Research Institute and School of Dentistry, University of California, Los Angeles, CA, United States of America.
authors.forename: Albert
authors.initials: A
authors.lastname: Ha
authors.affiliation: Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.
authors.forename: Kazunari K
authors.initials: KK
authors.lastname: Yokoyama
authors.affiliation: Dental and Craniofacial Research Institute and School of Dentistry, University of California, Los Angeles, CA, United States of America; Surgical Oncology & Jonsson Comprehensive Cancer Center, University of California, Los Angeles, CA, United States of America.
authors.forename: Robert H
authors.initials: RH
authors.lastname: Chiu
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000502:physiology; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D003513:Cycloheximide / Q000494:pharmacology; D021984:Cyclophilin A / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D016572:Cyclosporine / Q000494:pharmacology; D024202:Electrophoretic Mobility Shift Assay; D004789:Enzyme Activation; D005347:Fibroblasts; D015870:Gene Expression; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D007375:Interleukin-1 / Q000096:biosynthesis; D015850:Interleukin-6 / Q000096:biosynthesis; D016209:Interleukin-8 / Q000096:biosynthesis; D007783:Lactones / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008822:Mice, Transgenic; D011485:Protein Binding; D015398:Signal Transduction; D013141:Spiro Compounds / Q000494:pharmacology; D051996:Transcription Factor RelA / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation
chemical_list: D007375:Interleukin-1; D015850:Interleukin-6; D016209:Interleukin-8; D007783:Lactones; C498144:Rela protein, mouse; D013141:Spiro Compounds; D051996:Transcription Factor RelA; C121329:sanglifehrin A; D016572:Cyclosporine; D003513:Cycloheximide; D021984:Cyclophilin A