Enhanced phosphorylation of STAT-1 is dependent on double-stranded RNA-dependent protein kinase signaling in HLA-B27-expressing U937 monocytic cells

Objective To study the phosphorylation of STAT-1 in HLAB27transfected human monocytic cells and the role of the signaling molecules double-stranded RNAdependent 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-B27positive monocytic cells when compared to control cells, even prior to stimulation with LPS or bacteria. This phenotype was associated with the expression of HLAB27 heavy chains that misfold. In addition, phosphorylation of STAT-1 was dependent on PKR. Conclusion. 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.