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. 2006 Aug 1;108(3):821-9.
doi: 10.1182/blood-2005-11-006817.

RAX, the PKR activator, sensitizes cells to inflammatory cytokines, serum withdrawal, chemotherapy, and viral infection

Richard L Bennett  1 , William L Blalock, Dean M Abtahi, Yu Pan, Sue A Moyer, W Stratford May
Affiliations

RAX, the PKR activator, sensitizes cells to inflammatory cytokines, serum withdrawal, chemotherapy, and viral infection

Richard L Bennett et al. Blood. .

Abstract (VSports注册入口)

While the interferon (IFN)-inducible double-stranded RNA (dsRNA)-dependent protein kinase PKR is reported to initiate apoptosis in some instances, the mechanism by which diverse stress stimuli activate PKR remains unknown. Now we report that RAX, the only known cellular activator for PKR, initiates PKR activation in response to a broad range of stresses including serum deprivation, cytotoxic cytokine or chemotherapy treatment, or viral infection. Thus, knock-down of RAX expression by 80% using small interfering RNA (siRNA) prevents IFNgamma/tumor necrosis factor alpha (TNFalpha)-induced PKR activation and eIF2alpha phosphorylation, IkappaB degradation, IRF-1 expression, and STAT1 phosphorylation, resulting in enhanced murine embryonic fibroblast (MEF) cell survival. In contrast, expression of exogenous RAX, but not of the nonphosphorylatable, dominant-negative RAX(S18A) mutant, sensitizes cells to IFNgamma/TNFalpha, mitomycin C (MMC), or serum deprivation in association with increased PKR activity and apoptosis. Furthermore, RAX(S18A) expression in Fanconi anemia complementation group C-null MEF cells not only prevents PKR activation but also blocks hypersensitivity to IFNgamma/TNFalpha or mitomycin C that results in enhanced apoptosis. In addition, reduced RAX expression facilitates productive viral infection with vesicular stomatitis virus (VSV) and promotes anchorage-independent colony growth of MEF cells. Collectively, these data indicate that RAX may function as a negative regulator of growth that is required to activate PKR in response to a broad range of apoptosis-inducing stress VSports手机版. .

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Figures

Figure 1.
Figure 1.
Expression of siRNA to RAX promotes cell survival in medium with low serum. (A) Western blots using cell lysates from MEFs expressing the indicated siRNAs as well as from cell lines that express exogenous RAX or RAX(S18A). (B) Growth rate of MEF cell lines under serum-replete (10%) conditions. (C) Viability measured by TUNEL assay in medium with low (0.1%) serum of cell lines with reduced RAX levels caused by siRNA expression or expressing either exogenous RAX or RAX(S18A).
Figure 2.
Figure 2.
Reduced levels of RAX prevent IFNγ/TNFα-induced apoptosis. (A) IFNγ up-regulates PKR but not RAX in MEF cell lines. Western blots using antibodies to PKR or RAX with lysates from cells treated with 10 ng/mL IFNγ and TNFα. (B) Viability of MEF cells expressing either exogenous RAX or RAX(S18A) compared with vector control cells after treatment with 10 ng/mL IFNγ and/or 10 ng/mL TNFα for 24 hours. (C) Viability of MEFs expressing RAX, RAX(S18A), or reduced RAX that were pretreated with 20 ng/mL IFNγ for 24 hours followed by increasing concentrations of TNFα for 24 hours measured by TUNEL assay. (D) Relative PKR activity was assessed by autophosphorylation assay. Results represent the averaging of 3 separate experiments that quantify activated PKR and total PKR by autoradiography and Western blotting. Cells were treated with 10 ng/mL IFNγ for 24 hours followed by addition of 10 ng/mL TNFα for the indicated times. (E) Relative eIF2α phosphorylation determined by quantifying Western blots from 3 separate experiments using antibody to eIF2α and phosphoserine-51 eIF2α following treatment with 10 ng/mL IFNγ for 24 hours and with 10 ng/mL TNFα for the indicated times.
Figure 3.
Figure 3.
Regulators of transcription and survival are affected by RAX expression during IFNγ/TNFα treatment. (A) Western blotting was used to monitor IκBα degradation and IRF-1 production after cells had been treated with 10 ng/mL IFNγ followed by treatment with 10 ng/mL TNFα for the indicated times. (B) The level of STAT1 and phosphoserine 727 STAT1 were monitored after IFNγ/TNFα treatment by Western blotting.
Figure 4.
Figure 4.
Hypersensitivity of Fancc–/– cells to IFNγ/TNFα or mitomycin C can be suppressed by expression of the dominant negative RAX(S18A) mutant. (A) Western blots with lysates from Fancc–/– MEF cells expressing either RAX or RAX(S18A). (B) Survival as determined using TUNEL assay after treatment with 10 ng/mL IFNγ and/or 10 ng/mL TNFα of Fancc–/– cell lines. (C) Autoradiographs and Western blots from 3 separate PKR autophosphorylation assays were quantified and used to graph the increase in active PKR relative to total PKR protein level after treatment with 10 ng/mL IFNγ for 24 hours followed by treatment with 10 ng/mL TNFα for the indicated times. (D) Survival of normal MEF and Fancc–/– MEF cell lines expressing either exogenous RAX or RAX(S18A) after 24 hours of treatment with the indicated concentrations of mitomycin C. Viability was measured using TUNEL assay.
Figure 5.
Figure 5.
RAX deficiency causes failure of host antiviral defense to VSV infection. (A) Viability of MEF cell lines, as measured by trypan blue dye exclusion, 24 hours after infection with VSV at the indicated multiplicity of infection (MOI). (B) The increase in PKR autophosphorylation activity relative to total PKR level after the indicated time of infection with VSV at an MOI of 1 was determined by densitometry analysis of autoradiographs and Western blots from 3 separate experiments as described and graphed. (C) Densitometry analysis of Western blots from 3 separate experiments using antibodies specific for eIF2α and phosphoserine-51 eIF2α was used to determine and graph the fold increase in phospho-eIF2α relative to total eIF2α at the indicated times after VSV infection at an MOI of 1. (D) Western blotting with antibody R1 was used to monitor viral protein production of N, P, and M proteins at the indicated times after infection. (E) Virus titers of supernatants from infected cells 24 hours after infection were measured by plaque assay using BHK-21 cells. pfu indicates plaque-forming unit.
Figure 6.
Figure 6.
Reduced levels of RAX or expression of RAX(S18A) promotes anchorage-independent colony formation. MEF cells with reduced RAX or expressing exogenous RAX or RAX(S18A) were assayed for colony formation after 14 days of growth in soft agar. A representative crystal violet–stained plate for each cell line is shown. The transformation efficiency for each cell line was determined by averaging the number of colonies formed (groups of approximately 50 or more cells) from 4 separate plates divided by the total number of cells plated (1 × 104) ± SD.
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