title: Enhanced phosphorylation of STAT-1 is dependent on double-stranded RNA-dependent protein kinase signaling in HLA-B27-expressing U937 monocytic cells.
abstract: OBJECTIVE
  To study the phosphorylation of STAT-1 in HLA-B27-transfected human monocytic cells and the role of the signaling molecules double-stranded RNA-dependent protein kinase (PKR) and p38 in STAT-1 phosphorylation.
  
  
  METHODS
  U937 human monocytic cell transfectants stably expressing wild-type HLA-B27 or mutated HLA-B27 heavy chains with amino acid substitutions in the B pocket were prepared. Mock-transfected cells were prepared using the antibiotic resistance vectors (pSV2neo or RSV5neo) alone. Phorbol myristate acetate-differentiated cells were stimulated with lipopolysaccharide (LPS) or infected with Salmonella enteritidis. The phosphorylation and expression levels of STAT-1 protein were detected by Western blotting and flow cytometry. Specific inhibitors were added in cell culture to study the role of PKR and p38 in STAT-1 phosphorylation.
  
  
  RESULTS
  STAT-1 was constitutively highly phosphorylated on the tyrosine 701 residue in HLA-B27-positive monocytic cells when compared to control cells, even prior to stimulation with LPS or bacteria. This phenotype was associated with the expression of HLA-B27 heavy chains that misfold. In addition, phosphorylation of STAT-1 was dependent on PKR.
  
  
  CONCLUSIONS
  Our results show that STAT-1 tyrosine 701 is constitutively highly phosphorylated in the HLA-B27-expressing monocyte/macrophage cell line. Since phosphorylation of tyrosine 701 on STAT-1 is sufficient to induce interferon (IFN)-dependent genes, constitutive activity of this phosphorylation site may lead to the overexpression of IFN-dependent genes, as well as other STAT-1-dependent genes, in HLA-B27 monocyte/macrophages. Our results offer a mechanism by which B27 expression alone, without any external trigger, is potentially capable of inducing activation of STAT-1, a critical regulator of the inflammatory response.
authors.affiliation: National Institute for Health and Welfare, Turku, Finland. marja.ruuska@utu.fi
authors.forename: Marja
authors.initials: M
authors.lastname: Ruuska
authors.forename: Anna S
authors.initials: AS
authors.lastname: Sahlberg
authors.forename: Robert A
authors.initials: RA
authors.lastname: Colbert
authors.forename: Kaisa
authors.initials: K
authors.lastname: Granfors
authors.forename: Markus A
authors.initials: MA
authors.lastname: Penttinen
mesh_terms: D005786:Gene Expression Regulation / Q000187:drug effects; D020868:Gene Silencing; D015796:HLA-B27 Antigen / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054884:Host-Pathogen Interactions; D006801:Humans; D007372:Interferons / Q000235:genetics; D008070:Lipopolysaccharides / Q000494:pharmacology; D009000:Monocytes / Q000276:immunology / Q000378:metabolism* / Q000382:microbiology; D009154:Mutation; D010766:Phosphorylation; D011487:Protein Conformation; D017510:Protein Folding; D050794:STAT1 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D012477:Salmonella enteritidis / Q000187:drug effects / Q000276:immunology; D015398:Signal Transduction; D020298:U937 Cells; D019892:eIF-2 Kinase / Q000235:genetics / Q000378:metabolism*
chemical_list: D015796:HLA-B27 Antigen; D008070:Lipopolysaccharides; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D007372:Interferons; D019892:eIF-2 Kinase