Role of protein kinase R in double-stranded RNA-induced expression of nitric oxide synthase in human astroglia

Abstract

Environmental factor(s), such as viral infection, has been implicated as one of the triggering events leading to neuroinflammation in multiple sclerosis. This study underlines the importance of double-stranded RNA (dsRNA), the active component of a viral infection, in inducing the expression of inducible nitric oxide synthase (iNOS) in human astroglia. DsRNA in the form of synthetic polyinosinic-polycytidylic acid (poly IC) induced expression of iNOS and iNOS promoter-driven luciferase activity through activation of nuclear factor (NF)-kappaB and CCAAT/enhancer-binding proteinbeta (C/EBPbeta). In addition, we show that inhibitors of protein kinase R attenuated iNOS by suppressing the activation of NF-kappaB but not C/EBPbeta. In contrast, knock down of p38 mitogen-activated protein kinase (MAPK) attenuated iNOS by suppressing the activation of C/EBPbeta but not NF-kappaB. This study delineates a novel role of dsRNA in inducing the expression of iNOS through dsRNA-activated protein kinase (PKR)-mediated activation of NF-kappaB and p38-mediated activation of C/EBPbeta in human astroglia that may participate in virus-induced neurological abnormalities.

Figure 1

Dose‐dependent induction of iNOS by poly IC in human astrocytes. U373MG astroglial cells were stimulated with different concentrations of poly IC under serum‐free condition. A: After 24 h, supernatants were used for nitrite assay. B: Cell homogenates were immunoblotted with antibodies against mouse macrophage iNOS. C: After 6 h of incubation, Northern blot analysis for iNOS mRNA was carried out. Human iNOS promoter‐driven luciferase activity was assayed in U373MG astroglial cells (D) and primary astrocytes (E) as described in Section 2. Data are means±S.D. of three different experiments.

Figure 2

Activation of NF‐κB in poly IC‐treated U373MG astroglial cells. A: After different minutes of stimulation with 100 μg/ml of poly IC under serum‐free condition, nuclear proteins were examined for EMSA. The upper arrow indicates the induced NF‐κB band, and the lower arrow indicates the unbound probe. B: Transcriptional activity of NF‐κB was assayed as described in Section 2.

Figure 3

Activation of C/EBPβ in poly IC‐treated U373MG astroglial cells. A: At different minutes of stimulation with 100 μg/ml of poly IC, cells were taken out for EMSA. B: Nuclear proteins were examined for EMSA using wild‐type and mutated C/EBP probes. C: 1 μg of antibodies against C/EBPβ or a non‐specific protein were included in the binding reaction for supershift analysis. The upper, middle and lower arrows indicate the induced DNA‐binding activity of C/EBPβ, the induced DNA‐binding activity of another C/EBP isoform and the unbound probes respectively. D: Transcriptional activity of C/EBPβ was assayed as described above.

Figure 4

Effect of ΔPKR and Δp38 on iNOS promoter‐driven luciferase activity (A) and activation of NF‐κB (B) and C/EBPβ (C) in poly IC‐stimulated U373MG astroglial cells. A: Cells were cotransfected with 0.5 μg of phiNOS‐Luc, 50 ng of pRL‐TK and 0.5 μg of either ΔPKR, Δp38 or empty vector (pcDNA3). After 24 h of transfection, cells were stimulated with 100 μg/ml of poly IC for 12 h. Cells were cotransfected with 0.5 μg of either ΔPKR, Δp38 or empty vector, 50 ng of pRL‐TK and 0.5 μg of either pNF‐κB‐Luc (B) or pC/EBPβ‐Luc (C). After 24 h of transfection, cells were stimulated with 100 μg/ml of poly IC for 6 h. Firefly and Renilla luciferase activities were analyzed. *P<0.01 versus empty vector‐transfected cells.

Figure 5

Effect of pMT‐K3L on iNOS promoter‐driven luciferase activity (A) and the activation of NF‐κB (B) and C/EBPβ (C) in poly IC‐stimulated U373MG astroglial cells. A: Cells were cotransfected with 0.5 μg of either pMT‐K3L or empty vector (pMT), 0.5 μg of phiNOS‐Luc and 50 ng of pRL‐TK. After 24 h of transfection, cells were stimulated with 100 μg/ml of poly IC for 12 h. Cells were cotransfected with 0.5 μg of either pMT‐K3L or empty vector, 50 ng of pRL‐TK and 0.5 μg of either pNF‐κB‐Luc (B) or pC/EBPβ‐Luc (C). After 24 h of transfection, cells were stimulated with 100 μg/ml of poly IC for 6 h. Firefly and Renilla luciferase activities were analyzed. *P<0.01 versus empty vector‐transfected cells.