Involvement of Protein Kinase R in Double-Stranded RNA-Induced Proteasomal Degradation of Hypoxia Inducible Factor-1α

Abstract

Hypoxia inducible factor-1α (HIF-1α) is a crucial therapeutic target in various diseases, including cancer and fibrosis. We previously demonstrated that transfection with double-stranded RNA (dsRNA), including polyI:C and the dsRNA genome of mammalian orthoreovirus, resulted in significant reduction in HIF-1α protein levels in cultured cells; however, it remained to be elucidated how dsRNA induced down-regulation of HIF-1α protein levels. In this study, we examined the mechanism of dsRNA-mediated down-regulation of HIF-1α protein levels. We found that among the various cellular factors involved in dsRNA-mediated innate immunity, knockdown and knockout of protein kinase R (PKR) significantly restored HIF-1α protein levels in dsRNA-transfected cells, indicating that PKR was involved in dsRNA-mediated down-regulation of HIF-1α. Proteasome inhibitors significantly restored the HIF-1α protein levels in dsRNA-transfected cells. Ubiquitination levels of HIF-1α were increased by transfection with dsRNA. These findings indicated that degradation of HIF-1α in a ubiquitin-proteasome pathway was promoted in a PKR-dependent manner following dsRNA transfection. Expression of not only HIF-1α but also several proteins, including CDK4 and HER2, was down-regulated following dsRNA transfection. These data provide important clues for elucidation of the mechanism of dsRNA-mediated cellular toxicity, as well as for therapeutic application of dsRNA.

Keywords: Double-stranded RNA; HIF-1α; PKR; Proteasome; Ubiquitin.

Fig. 1

Involvement of PKR in dsRNA-mediated down-regulation of HIF-1α. A Knockdown efficiencies of siRNAs against dsRNA-mediated innate immunity-related genes. H1299 cells were transfected with control siRNA (open bar) and siRNAs against dsRNA-mediated innate immunity-related genes (closed bar) at 50 nM. Knockdown efficiencies were determined 24 h after transfection by real-time RT-PCR analysis. B HIF-1α expression levels in H1299 cells with knockdown of dsRNA-mediated innate immunity-related genes following transfection with the reovirus dsRNA genome. H1299 cells were transfected with siRNAs at 50 nM for 24 h, followed by transfection with the reovirus dsRNA genome at 4 ng/ml for 24 h. C quantitation of results shown in B and independent replicates (n = 3-4). Statistical significance relative to mock cells pretreated with control siRNA was tested by paired t test. *p < 0.05. Data are expressed as means ± S.D. (n = 3-4). D Knockdown efficiencies of siRNAs against PKR. H1299 cells were transfected with control siRNA and siRNAs against PKR at 50 nM. Knockdown efficiencies were determined as described above. E Restoration of HIF-1α expression in reovirus dsRNA genome-transfected H1299 cells by PKR knockdown using PKR-targeted siRNAs with different sequences. F Restoration of HIF-1α expression in reovirus dsRNA genome-transfected H1299 PKR-KO cells. H1299 PKR-KO cells were transfected with the reovirus dsRNA genome at 4 ng/ml for 24 h. G quantitation of results shown in F and independent replicates (n = 5). Statistical significance relative to mock cells was tested by paired t test. *p < 0.05. Data are expressed as means ± S.D. (n = 5). The representative images from at least three independent experiments are shown.

Fig. 2

Kinase activity of PKR is necessary for dsRNA-mediated down-regulation of HIF-1α. A dsRNA-mediated down-regulation of HIF-1α in the presence of a PKR inhibitor, 2-AP. H1299 cells were pretreated with 2-AP at 10 mM for 2 h, followed by transfection with the reovirus dsRNA genome at 4 ng/ml for 24 h. B Knockdown efficiency of an siRNA against eIF2 in H1299 cells. H1299 cells were transfected with an siRNA against eIF2α at 50 nM for 24 h, followed by real-time RT-PCR analysis. C HIF-1α expression levels in eIF2α-knocked down cells following transfection with dsRNA. H1299 cells were transfected with an siRNA against eIF2α for 24 h, followed by transfection with the reovirus dsRNA genome at 4 ng/ml for 24 h. Data are expressed as means ± S.D. (n = 4). The representative images from at least two independent experiments are shown.

Fig. 3

Induction of proteasomal degradation of HIF-1α in dsRNA-transfected cells. A Restoration of HIF-1α protein levels in dsRNA-transfected cells by pre-treatment with proteasome inhibitors. H1299 cells were pretreated with MG-132 and epoxomicin for 30 min, followed by transfection with the reovirus dsRNA genome at 4 ng/ml for 24 h. B Ubiquitination levels of HIF-1α following transfection with dsRNA. H1299 cells were transfected with the reovirus dsRNA genome at 4 ng/ml for 24 h. Ubiquitinated proteins were immune-precipitated, followed by western blotting analysis using anti-HIF-1 antibody. The representative images from at least two independent experiments are shown.

Fig. 4

Induction of proteasomal degradation of CDK4, AKT, and HER2 following transfection with dsRNA. A Expression levels of CDK4, AKT, and HER2 in human tumor cells following transfection with dsRNA. Cells were pretreated with DMSO or epoxomicin for 30 min, followed by transfection with the reovirus dsRNA genome at 4 ng/ml for 24 h. DMSO, dimethyl sulfoxide; Epo, Epoxomicin. B Restoration of CDK4 expression by knockdown of PKR in dsRNA-transfected cells. H1299 cells were transfected with an siRNA against PKR for 24 h, followed by transfection with the reovirus dsRNA genome at 4 ng/ml for 24 h. The representative images from at least two independent experiments are shown.