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 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.

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.