Protein Kinase R Mediates the Inflammatory Response Induced by Hyperosmotic Stress

ABSTRACT

High extracellular osmolarity results in a switch from an adaptive to an inflammatory gene expression program. We show that hyperosmotic stress activates the protein kinase R (PKR) independently of its RNA-binding domain. In turn, PKR stimulates nuclear accumulation of nuclear factor κB (NF-κB) p65 species phosphorylated at serine-536, which is paralleled by the induction of a subset of inflammatory NF-κB p65-responsive genes, including inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6), and IL-1β. The PKR-mediated hyperinduction of iNOS decreases cell survival in mouse embryonic fibroblasts via mechanisms involving nitric oxide (NO) synthesis and posttranslational modification of proteins. Moreover, we demonstrate that the PKR inhibitor C16 ameliorates both iNOS amplification and disease-induced phenotypic breakdown of the intestinal epithelial barrier caused by an increase in extracellular osmolarity induced by dextran sodium sulfate (DSS) in vivo. Collectively, these findings indicate that PKR activation is an essential part of the molecular switch from adaptation to inflammation in response to hyperosmotic stress.

KEYWORDS:

• DSS
• PKR
• hyperosmotic stress
• iNOS

ACKNOWLEDGMENTS

We thank Paul Fox for the donation of GAPDH-HA plasmids and Josephine Kam, Tai Dermawan, and George Stark for critical discussion of the manuscript. We also graciously thank Gokhan Hotamisligil for the generous gift of PKR-deficient and -reconstituted cell lines.

Author contributions are as follows. K.T.F., M.M., D.K., X.-H.G., A.E.K., and M.H. conceived and designed the experiments. K.T.F., M.M., B.-J.G., R.J., J.W., D.K., X.-H.G., A.S., P.R., and M.B. performed the experiments. K.T.F., M.M., B.-J.G., R.J., D.K., X.-H.G., M.L., P.R., and M.H. analyzed the data. M.L., E.D.C., M.D., P.R., and M.H. provided reagents/materials/analysis tools. K.T.F. and M.H. wrote the paper, with edits by B.-J.G., D.K., and P.R.

We declare that we have no competing financial interests.