Requirement for protein kinase R in interleukin-1alpha-stimulated effects in cartilage.
Interleukin-1 (IL-1) has pleiotropic effects in cartilage. The interferon-induced, double stranded RNA-activated protein kinase PKR that phosphorylates eukaryotic initiation factor 2 (eIF2) alpha has been implicated in cytokine effects in chondrocytes. A compound was recently identified that potently suppresses PKR autophosphorylation (IC50 approximately 200 etaM) and partially restores PKR-inhibited translation in a cell-free system with significant effect in the nanomolar range. The objectives of this study were to exploit this potent PKR inhibitor to assess whether PKR kinase activity is required for catabolic and proinflammatory effects of IL-1alpha in cartilage and to determine whether IL-1alpha causes an increase in eIF2alpha phosphorylation that is antagonized by the PKR inhibitor. Cartilage explants were incubated with the PKR inhibitor and IL-1alpha. Culture media were assessed for sulfated glycosaminoglycan as an indicator of proteoglycan degradation and for prostaglandin E(2). Cartilage extracts were analyzed by Western blot for cyclooxygenase-2 and phosphorylated signaling molecules. Nanomolar concentrations of the PKR inhibitor suppressed proteoglycan degradation and cyclooxygenase-2 accumulation in IL-1alpha-activated cartilage. The PKR inhibitor stimulated or inhibited PGE(2) production with a biphasic dose response relationship. IL-1alpha increased the phosphorylation of both PKR and eIF2alpha, and nanomolar concentrations of PKR inhibitor suppressed the IL-1alpha-induced changes in phosphorylation. The results strongly support PKR involvement in pathways activated by IL-1alpha in chondrocytes.
Tam
Christine L
CL
Department of Orthopaedic Surgery, Massachusetts General Hospital, Boston, MA 02114-2696, United States.
Hofbauer
Maria
M
Towle
Christine A
CA
D000818:Animals; D015153:Blotting, Western; D050824:Carpal Joints / Q000187:drug effects / Q000378:metabolism; D002356:Cartilage / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D002417:Cattle; D019902:Chondrocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D017077:Culture Media, Conditioned / Q000032:analysis / Q000737:chemistry; D051546:Cyclooxygenase 2 / Q000378:metabolism; D004229:Dithiothreitol / Q000031:analogs & derivatives / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism; D006025:Glycosaminoglycans / Q000378:metabolism; D006801:Humans; D018836:Inflammation Mediators / Q000378:metabolism; D053582:Interleukin-1alpha / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011509:Proteoglycans / Q000378:metabolism; D013431:Sulfates / Q000378:metabolism; D013997:Time Factors; D019892:eIF-2 Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*
D017077:Culture Media, Conditioned; D015852:Eukaryotic Initiation Factor-2; D006025:Glycosaminoglycans; D018836:Inflammation Mediators; D053582:Interleukin-1alpha; D047428:Protein Kinase Inhibitors; D011509:Proteoglycans; D013431:Sulfates; C085285:dithiothreitol tetraacetate; D051546:Cyclooxygenase 2; D019892:eIF-2 Kinase; D004229:Dithiothreitol