Activation of the Double‐Stranded‐RNA‐Activated Protein Kinase and Induction of Vascular Cell Adhesion Molecule‐1 by Poly (I) · Poly (C) in Endothelial Cells

Abstract

Double‐stranded RNA (dsRNA) induces the vascular cell adhesion molecule VCAM‐1 to high levels of expression in human umbilical vein endothelial (HUVE) cells. Although VCAM‐1 is also induced by the cytokine interleukin 1β (IL‐1β), activation of the dsRNA‐activated protein kinase (PKR) occurs only in response to incubation with dsRNA but not with IL‐1β. Incubation of HUVE cells with the synthetic dsRNA, poly (I) · poly (C), activates PKR with increased autophosphorylation, increased phosphorylation of the translation factor eIF2α, and increased activation of the transcription factor NF‐кB. Promoter analysis in HUVE cells using a VCAM‐1 promoter linked to CAT reporter gene demonstrates that poly (I) · poly (C) responsiveness resides in the minimal VCAM‐1 promoter that contains two NF‐кB sites, and deletion of the NF‐кB sites eliminates basal and poly (I) · poly (C)‐induced CAT activity, supporting the importance of NF‐кB in the poly (I) · poly (C)‐mediated induction of VCAM‐1. In vitro studies using purified reagents demonstrate that PKR is capable of phosphorylating IкBα (the inhibitory subunit of NF‐кB) in a dsRNA‐dependent manner. This suggests that phosphorylation of IкBα by PKR could be an initial step in the activation of NF‐кB by dsRNA. NF‐кB is also activated by IL‐1β in HUVE cells, but this activation occurs without increased PKR autophosphorylation or eIF2α phosphorylation. Poly (I) · poly (C) induces VCAM‐1 mRNA levels that are dramatically higher and sustained longer than levels induced by IL‐1β. Although phosphorylation of eIF2α interferes with protein translation, sufficient VCAM‐1 mRNA translation occurs in response to poly (I) · poly (C) to yield VCAM‐1 protein levels that are similar to levels that are induced by IL‐1bT. This suggests that the higher, sustained VCAM‐1 mRNA levels that occur in response to incubation with poly (I) · poly (C) compensate for the partial translational block resulting from increased eIF2α phosphorylation. These studies indicate that transcrip‐tional and translational regulatory events that occur in response to activation of PKR by dsRNA are important in the regulation of VCAM‐1 gene expression in HUVE cells.

Neoplastic progression in melanoma and colon cancer is associated with increased expression and activity of the interferon-inducible protein kinase, PKR

“…The IKK complex then phosphorylates IKB, the inhibitory subunit of NFkB. This event leads to IkB degradation with subsequent release and nuclear translocation of NFkB (Bonnet et al, 2000;Gil et al, , 2001Ishii et al, 2001;Offermann et al, 1995;Zamanian-Daryoush et al, 2000). It has been postulated that increasing PKR activity has a net tumor suppressive effect within the cell.…”

IFN-α Sensitizes Human Umbilical Vein Endothelial Cells to Apoptosis Induced by Double-Stranded RNA

“…For example, dsRNA induces the expression of the cellular adhesion molecules ICAM-1, VCAM-1, and E-selectin (26,27), molecules that are important in adhesion and transendothelial migration of leukocytes that could help contain the viral infection (28,29). dsRNA also induces expression of MHC I (19), a protein critical for the presentation of foreign Ags to CTL, contributing to the acquired immune response (30,31).…”

“…Endothelial cells also produce the cytokines IL-1␣, IL-1␤, IL-6, and type I IFN in response to dsRNA (26,32,33), thereby contributing to leukocyte maturation and alerting neighboring cells to the potential risk of viral infection. The dsRNA also reduces protein synthesis, most likely through the activation of the antiviral proteins PKR and 2Ј5Ј-OAS (26,27). This restricts the expression of viral proteins, making it more difficult for the virus to make progeny virions.…”

“…Although endothelial cells are known to respond to dsRNA with multiple changes in gene expression and a reduction in protein synthesis (19,26,27), the role of apoptosis in the endothelial response to synthetic or viral dsRNA has not been reported. In this study, we demonstrate that multiple caspases are activated by dsRNA, and IFN-␣ priming dramatically accelerates their activation by dsRNA.…”

Human breast cancer cells contain elevated levels and activity of the protein kinase, PKR

“…Phosphorylated eIF2 entraps another initiation factor, eIF2B, in an inactive complex, which subsequently causes inhibition of translation (for review: Clemens and Elia, 1997). PKR also phosphorylates other proteins that are involved in the transcription pathway such as, IkB, the inhibitor of transcription factor, NF-kB (Kumar et al, 1997;Maran et al, 1994;O ermann et al, 1995), NF90 (Langland et al, 1999;Patel et al, 1999;Parker and Mathews, unpublished data), RNA helicase A (Parker and Mathews, unpublished data) and HIV-1 Tat .…”