PKR Inhibitor Research Articles

242 : RNase L induces autophagy via C-Jun N-terminal kinase and double-stranded RNA-dependent protein kinase signaling pathway

Autophagy is a tightly regulated mechanism that mediates sequestration, degradation and recycling of cellular proteins, organelles and pathogens. Several proteins associated with autophagy regulate host responses to viral infections. Ribonuclease L (RNase L) is activated during viral infections and cleaves cellular and viral single-stranded RNAs, including rRNAs in ribosomes. We demonstrate that direct activation of RNase L coordinates the activation of c-Jun N-terminal kinase (JNK) and double-stranded RNA-dependent protein kinase (PKR) to induce autophagy with hallmarks as accumulation of autophagic vacuoles, p62(SQSTM1) degradation and conversion of Microtubule-associated Protein Light Chain 3-I (LC3-I) to LC3-II. Accordingly, treatment of cells with pharmacological inhibitors of JNK or PKR and mouse embryonic fibroblasts (MEFs) lacking JNK1/2 or PKR showed reduced autophagy levels. Furthermore, RNase L-induced JNK activity promoted Bcl-2 phosphorylation, disrupted the Beclin1-Bcl-2 complex and stimulated autophagy. Viral infection with Encephalomyocarditis virus (EMCV) or Sendai virus led to higher levels of autophagy in wild-type (WT) MEFs compared to RNase L knock out (KO) MEFs. Inhibition of RNase L-induced autophagy using Bafilomycin A1 or 3-methyladenine suppressed viral growth in initial stages; in later stages autophagy promoted viral replication dampening the antiviral effect. Induction of autophagy by activated RNase L is independent of the paracrine effects of interferon (IFN). Our findings suggest a novel role of RNase L in inducing autophagy affecting the outcomes of viral pathogenesis.

Altered Regulation of ELAVL1/HuR in HLA-B27–Expressing U937 Monocytic Cells

ObjectiveTo investigate the role of HLA-B27 expression in the regulation of RNA binding protein (RBP) Embryonic Lethal Abnormal Vision (ELAV) L1/Human antigen R (HuR) expression in Salmonella-infected or LPS-stimulated human monocytic cells, since HuR is a critical regulator of the post-transcriptional fate of many genes (e.g. TNFα) important in inflammatory response.MethodsU937 monocytic cells were stably transfected with pSV2neo resistant vector (mock), wild type HLA–B27, or mutated HLA–B27 with amino acid substitutions in the B pocket. Cells were differentiated, infected with Salmonella enteritidis or stimulated with lipopolysaccharide. The expression levels of HuR protein and cleavage products (CP1 and CP2) were detected by Western blotting and flow cytometry. Specific inhibitors were used to study the role of PKR and p38 in HuR expression and generation of CPs. TNFα and IL-10 secretion after p38 and PKR inhibition were measured by ELISA.ResultsFull length HuR is overexpressed and HuR cleavage is disturbed in U937 monocytic cells expressing HLA-B27 heavy chains (HC). Increased full length HuR expression, disturbed cleavage and reduced dependence on PKR after infection correlate with the expression of glutamic acid 45 in the B pocket that is linked to the misfolding of HLA-B27.ConclusionResults show that the expression of HLA-B27 HCs modulates the intracellular environment of U937 monocyte/macrophages by altering HuR regulation. This phenomenon is at least partly dependent on the misfolding feature of the B27 molecule. Since HuR is an important regulator of multiple genes involved in inflammatory response observations offer an explanation how HLA-B27 may modulate inflammatory response.

A deletion mutation of the gene P58IPK, the cellular inhibitor of PKR and PERK, results in a degenerative joint phenotype in mice

Searchable abstracts of presentations at key conferences on calcified tissues ISSN 2052-1219 (online)

PKR regulates proliferation, differentiation, and survival of murine hematopoietic stem/progenitor cells

AbstractProtein kinase R (PKR) is an interferon (IFN)-inducible, double-stranded RNA-activated kinase that initiates apoptosis in response to cellular stress. To determine the role of PKR in hematopoiesis, we developed transgenic mouse models that express either human PKR (TgPKR) or a dominant-negative PKR (TgDNPKR) mutant specifically in hematopoietic tissues. Significantly, peripheral blood counts from TgPKR mice decrease with age in association with dysplastic marrow changes. TgPKR mice have reduced colony-forming capacity and the colonies also are more sensitive to hematopoietic stresses. Furthermore, TgPKR mice have fewer hematopoietic stem/progenitor cells (HSPCs), and the percentage of quiescent (G0) HSPCs is increased. Importantly, treatment of TgPKR bone marrow (BM) with a PKR inhibitor specifically rescues sensitivity to growth factor deprivation. In contrast, marrow from PKR knockout (PKRKO) mice has increased potential for colony formation and HSPCs are more actively proliferating and resistant to stress. Significantly, TgPKR HSPCs have increased expression of p21 and IFN regulatory factor, whereas cells from PKRKO mice display mechanisms indicative of proliferation such as reduced eukaryotic initiation factor 2α phosphorylation, increased extracellular signal-regulated protein kinases 1 and 2 phosphorylation, and increased CDK2 expression. Collectively, data reveal that PKR is an unrecognized but important regulator of HSPC cell fate and may play a role in the pathogenesis of BM failure.

Abstract 5673: Novel inhibitors of the interferon-inducible, dsRNA-activated kinase PKR for myelodysplastic syndrome.

Abstract In hematopoietic stem/progenitor cells, the interferon-inducible, dsRNA-activated protein kinase, PKR, is a central mediator for the anti-proliferative effects of a wide variety of hematopoietic, inflammatory or cytotoxic stresses such as deprivation of IL-3, IL-5 or GM-CSF from hematopoietic factor-dependent cells. Once activated, PKR inhibits protein synthesis by phosphorylation of eIF2alpha, inhibits mitochondrial function by activating PP2A-dependent Bcl2 dephosphorylation, and activates signaling pathways such as NF-kappaB, p53 and STAT1 to regulate proliferation and apoptosis in response to cellular stress. Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematologic malignancies characterized by peripheral cytopenias, a hypercellular marrow with ineffective hematopoiesis and risk of transformation to acute myeloid leukemia (AML). Significantly, PKR is highly activated in myeloid progenitor cells from MDS patients and inhibition of PKR activity or siRNA-knockdown partially reverses the suppressive effects of IFN-gamma and TNF-alpha on hematopoietic colony formation using normal or MDS-derived CD34+ cells. Thus, increased PKR activity in hematopoietic stem cells may cooperate with driver/founder mutations to promote bone marrow failure and apoptosis characteristic of MDS, and inhibitors of PKR may be therapeutically useful to delay the onset and/or reduce the severity of MDS and its evolution to AML. Since there is only one commercially available pharmacologic PKR inhibitor that targets ATP binding and has been reported to have off target effects, we performed studies to identify novel small molecule compounds that specifically inhibit PKR-substrate association. Using the crystal structure of the PKR kinase domain-eIF2alpha complex, we employed a molecular docking strategy to screen in silico the NIH Developmental Therapeutic Program's repository of over 300,000 small molecules for compounds that interact with and inhibit PKR. The top 195 scoring small molecule compounds identified were obtained and validated in vitro using a LanthaScreen PKR kinase assay and 32P PKR assay. In addition, we screened the NIH/DTP Diversity and Challenge Compound Sets for inhibitors of PKR (2047 compounds total). Using these methods, we have identified 27 novel PKR inhibitors with an IC50 < 100 μM. Significantly, five are competitive inhibitors for eIF2alpha binding to PKR while the remaining inhibit ATP binding. These results identify a novel class of PKR inhibitors that specifically disrupt association of PKR with its substrate, eIF2alpha, and lay the groundwork for future studies that test whether these PKR inhibitors may be therapeutically useful to delay the onset of cytopenias and bone marrow failure associated with MDS and MDS evolution to AML. Citation Format: Richard L. Bennett, David A. Ostrov, W. Stratford May. Novel inhibitors of the interferon-inducible, dsRNA-activated kinase PKR for myelodysplastic syndrome. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5673. doi:10.1158/1538-7445.AM2013-5673

Abstract 4606: Loss of PKR promotes leukemia progression by activating Bcl-2 to inhibit apoptosis.

Abstract The interferon-inducible, dsRNA-dependent protein kinase, PKR, is activated by diverse cellular stresses. Loss of PKR expression or activity has been found in tumors including various types of leukemia, suggesting that loss of PKR may contribute to malignancy. However, no convincing conclusion has been reached regarding the relationship between PKR and leukemia progression. We analyzed 511 AML primary bone marrow samples by reverse phase protein array (RPPA) and discovered that PKR protein expression is > 50% reduced in CD34+ blasts from AML patients compared to CD34+ cells from healthy donors. This finding prompted us to further study how PKR affected progression of leukemia. PKR expression was either knocked down (by 82%) by specific SiRNA or directly inhibited by specific PKR inhibitor in leukemia cell lines. Significantly, inhibition of PKR decreased the rate of apoptosis following treatment with hydrogen peroxide (H2O2) or doxorubicin for 48 hours. In addition, bone marrow mononuclear cells form wild type, PKR null (-/-) or PKR transgenic mice were harvested and subjected to growth factor withdrawal for up to 120 hours. Cells isolated from PKR null mice had decreased apoptosis while cells from PKR transgenic mice had increased apoptosis compared to cells from wild type littermate mice. Further investigation showed that PKR inhibition led to reduction of PP2A activity, and a 2.4 fold increase in Bcl-2 phosphorylation. In addition, okadaic acid (an inhibitor of PP2A) had a similar effect to PKR inhibition on both Bcl-2 phosphorylation (1.74 fold increase) and stress-induced apoptosis. By contrast, treatment of cells with a PP2A activator, FTY 720, resulted in decreased Bcl-2 phosphorylation (by 2.24 fold) and enhanced apoptosis in response to H2O2. Furthermore, our study showed that following PKR inhibition, phosphorylation of Bcl-2 stabilized Bcl-2/Bax complex following H2O2 treatment, preventing Bax from being released, anchoring and perforating the mitochondrial outer membrane, which would initiate apoptotic process. To confirm our in vitro findings, a leukemia xenograft model was established in NSG mice using REH cells expressing luciferase. Mice receiving REH SiPKR cells displayed significantly faster tumor progression compared to mice receiving Sicontrol cells. Furthermore, tumors from REH SiPKR cells were more resistant to doxorubicin treatment compared to tumors from REH sicontrol cells. Taken together our results demonstrate that loss of PKR function inhibits PP2A-dependent Bcl-2 dephosphorylation resulting in increased leukemia progression and survival. In addition, since our studies indicate that PKR activation enhances the effectiveness of chemotherapy, PKR level/function status may serve as a clinical predictor of prognosis, disease relapse and resistance to chemotherapy. Citation Format: Xiaodong Cheng, Richard L. Bennett, Xiangfei Liu, Stratford May. Loss of PKR promotes leukemia progression by activating Bcl-2 to inhibit apoptosis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4606. doi:10.1158/1538-7445.AM2013-4606

Blocking the eIF2α Kinase (PKR) Enhances Positive and Negative Forms of Cortex-Dependent Taste Memory

Age-associated memory deterioration (and the decline in ability to acquire new information) is one of the major diseases of our era. Cognitive enhancement can be achieved by using psycho-stimulants, such as caffeine or nicotine, but very little is known about drugs that can enhance the consolidation phase of memories in the cortex, the brain structure considered to store, at least partially, long-term memories. We used cortex-dependent taste-learning paradigms to test the hypothesis that pharmacological manipulation of the translation initiation eIF2α, which plays a role in hippocampus-dependent memory, can enhance positive or negative forms of taste memories. We found that dephosphorylation (Ser51) of eIF2α, specifically in the cortex, is both correlated with and necessary for normal memory consolidation. To reduce eIF2α phosphorylation and improve memory consolidation, we pharmacologically inhibited one of the eIF2α kinases, PKR, which is known to be involved in brain aging and Alzheimer's disease. Systemic or local microinjection of PKR inhibitor to the gustatory cortex enhanced both positive and negative forms of taste memory in rats and mice. Our results provide clear evidence that PKR plays a major role in cortex-dependent memory consolidation and, therefore, that pharmacological inhibition of PKR is a potential target for drugs to enhance cognition.

A survey of host range genes in poxvirus genomes

Poxviruses are widespread pathogens, which display extremely different host ranges. Whereas some poxviruses, including variola virus, display narrow host ranges, others such as cowpox viruses naturally infect a wide range of mammals. The molecular basis for differences in host range are poorly understood but apparently depend on the successful manipulation of the host antiviral response. Some poxvirus genes have been shown to confer host tropism in experimental settings and are thus called host range factors. Identified host range genes include vaccinia virus K1L, K3L, E3L, B5R, C7L and SPI-1, cowpox virus CP77/CHOhr, ectromelia virus p28 and 022, and myxoma virus T2, T4, T5, 11L, 13L, 062R and 063R. These genes encode for ankyrin repeat-containing proteins, tumor necrosis factor receptor II homologs, apoptosis inhibitor T4-related proteins, Bcl-2-related proteins, pyrin domain-containing proteins, cellular serine protease inhibitors (serpins), short complement-like repeats containing proteins, KilA-N/RING domain-containing proteins, as well as inhibitors of the double-stranded RNA-activated protein kinase PKR. We conducted a systematic survey for the presence of known host range genes and closely related family members in poxvirus genomes, classified them into subgroups based on their phylogenetic relationship and correlated their presence with the poxvirus phylogeny. Common themes in the evolution of poxvirus host range genes are lineage-specific duplications and multiple independent inactivation events. Our analyses yield new insights into the evolution of poxvirus host range genes. Implications of our findings for poxvirus host range and virulence are discussed.

RNase L Induces Autophagy via c-Jun N-terminal Kinase and Double-stranded RNA-dependent Protein Kinase Signaling Pathways

Autophagy is a tightly regulated mechanism that mediates sequestration, degradation, and recycling of cellular proteins, organelles, and pathogens. Several proteins associated with autophagy regulate host responses to viral infections. Ribonuclease L (RNase L) is activated during viral infections and cleaves cellular and viral single-stranded RNAs, including rRNAs in ribosomes. Here we demonstrate that direct activation of RNase L coordinates the activation of c-Jun N-terminal kinase (JNK) and double-stranded RNA-dependent protein kinase (PKR) to induce autophagy with hallmarks as accumulation of autophagic vacuoles, p62(SQSTM1) degradation and conversion of Microtubule-associated Protein Light Chain 3-I (LC3-I) to LC3-II. Accordingly, treatment of cells with pharmacological inhibitors of JNK or PKR and mouse embryonic fibroblasts (MEFs) lacking JNK1/2 or PKR showed reduced autophagy levels. Furthermore, RNase L-induced JNK activity promoted Bcl-2 phosphorylation, disrupted the Beclin1-Bcl-2 complex and stimulated autophagy. Viral infection with Encephalomyocarditis virus (EMCV) or Sendai virus led to higher levels of autophagy in wild-type (WT) MEFs compared with RNase L knock out (KO) MEFs. Inhibition of RNase L-induced autophagy using Bafilomycin A1 or 3-methyladenine suppressed viral growth in initial stages; in later stages autophagy promoted viral replication dampening the antiviral effect. Induction of autophagy by activated RNase L is independent of the paracrine effects of interferon (IFN). Our findings suggest a novel role of RNase L in inducing autophagy affecting the outcomes of viral pathogenesis.

Double-stranded RNA-dependent protein kinase regulates insulin-stimulated chondrogenesis in mouse clonal chondrogenic cells, ATDC-5

Double-stranded RNA-dependent protein kinase (PKR) is an interferon-induced protein that has been identified and characterized as a translational inhibitor in an interferon-regulated antiviral pathway. PKR is also reported to play important roles in the regulation of cell growth and differentiation. We have previously demonstrated that PKR inactivation suppresses osteoblast calcification and osteoclast formation. However, reports concerning the roles of PKR in chondrogenesis are limited. In this study, we have demonstrated that PKR is required for the in vitro differentiation of the mouse clonal chondrogenic cell line ATDC-5. ATDC-5 cells treated with insulin differentiated into chondrocytes and produced an alcian-blue-positive cartilage matrix. The protein expression of signal transducers and activators of transcription (STAT) peaked at day 7 of differentiation, whereas the expression of SRY-box-containing gene 9 (Sox-9), which is a transcription factor for chondrocyte differentiation, increased gradually. When the cells were treated with a PKR inhibitor (2-aminopurine), the cartilage matrix formation decreased. The protein expression of STAT1 continued to increase up to day 21, whereas the expression of Sox-9 was low and did not increase. We also demonstrated that PKR was localized to a marginal region of the mandibular condyle cartilage in mouse embryos. Our findings suggest that PKR has important functions in the differentiation of chondrocytes through the modulation of STAT1 and Sox-9 expression.

The Herpes Simplex Virus 1 Us11 Protein Inhibits Autophagy through Its Interaction with the Protein Kinase PKR

Autophagy is now known to be an essential component of host innate and adaptive immunity. Several herpesviruses have developed various strategies to evade this antiviral host defense. Herpes simplex virus 1 (HSV-1) blocks autophagy in fibroblasts and in neurons, and the ICP34.5 protein is important for the resistance of HSV-1 to autophagy because of its interaction with the autophagy machinery protein Beclin 1. ICP34.5 also counteracts the shutoff of protein synthesis mediated by the double-stranded RNA (dsRNA)-dependent protein kinase PKR by inhibiting phosphorylation of the eukaryotic translation initiation factor 2α (eIF2α) in the PKR/eIF2α signaling pathway. Us11 is a late gene product of HSV-1, which is also able to preclude the host shutoff by direct inhibition of PKR. In the present study, we unveil a previously uncharacterized function of Us11 by demonstrating its antiautophagic activity. We show that the expression of Us11 is able to block autophagy and autophagosome formation in both HeLa cells and fibroblasts. Furthermore, immediate-early expression of Us11 by an ICP34.5 deletion mutant virus is sufficient to render the cells resistant to PKR-induced and virus-induced autophagy. PKR expression and the PKR binding domain of Us11 are required for the antiautophagic activity of Us11. However, unlike ICP34.5, Us11 did not interact with Beclin 1. We suggest that the inhibition of autophagy observed in cells infected with HSV-1 results from the activity of not only ICP34.5 on Beclin 1 but also Us11 by direct interaction with PKR.

DsRNA-Dependent Protein Kinase PKR and its Role in Stress, Signaling and HCV Infection

The double-stranded RNA-dependent protein kinase PKR plays multiple roles in cells, in response to different stress situations. As a member of the interferon (IFN)‑Stimulated Genes, PKR was initially recognized as an actor in the antiviral action of IFN, due to its ability to control translation, through phosphorylation, of the alpha subunit of eukaryotic initiation factor 2 (eIF2α). As such, PKR participates in the generation of stress granules, or autophagy and a number of viruses have designed strategies to inhibit its action. However, PKR deficient mice resist most viral infections, indicating that PKR may play other roles in the cell other than just acting as an antiviral agent. Indeed, PKR regulates several signaling pathways, either as an adapter protein and/or using its kinase activity. Here we review the role of PKR as an eIF2α kinase, its participation in the regulation of the NF-κB, p38MAPK and insulin pathways, and we focus on its role during infection with the hepatitis C virus (HCV). PKR binds the HCV IRES RNA, cooperates with some functions of the HCV core protein and may represent a target for NS5A or E2. Novel data points out for a role of PKR as a pro-HCV agent, both as an adapter protein and as an eIF2α-kinase, and in cooperation with the di-ubiquitin-like protein ISG15. Developing pharmaceutical inhibitors of PKR may help in resolving some viral infections as well as stress-related damages.

Double-Stranded RNA-Dependent Protein Kinase Regulates the Motility of Breast Cancer Cells

Double-stranded RNA (dsRNA)-dependent protein kinase (PKR) is an interferon-induced protein kinase that plays a central role in the anti-viral process. Due to its pro-apoptotic and anti-proliferative action, there is an increased interest in PKR modulation as an anti-tumor strategy. PKR is overexpressed in breast cancer cells; however, the role of PKR in breast cancer cells is unclear. The expression/activity of PKR appears inversely related to the aggressiveness of breast cancer cells. The current study investigated the role of PKR in the motility/migration of breast cancer cells. The activation of PKR by a synthesized dsRNA (PIC) significantly decreased the motility of several breast cancer cell lines (BT474, MDA-MB231 and SKBR3). PIC inhibited cell migration and blocked cell membrane ruffling without affecting cell viability. PIC also induced the reorganization of the actin cytoskeleton and impaired the formation of lamellipodia. These effects of PIC were reversed by the pretreatment of a selective PKR inhibitor. PIC also activated p38 mitogen-activated protein kinase (MAPK) and its downstream MAPK-activated protein kinase 2 (MK2). PIC-induced activation of p38 MAPK and MK2 was attenuated by the PKR inhibitor and the PKR siRNA, but a selective p38 MAPK inhibitor (SB203580) or other MAPK inhibitors did not affect PKR activity, indicating that PKR is upstream of p38 MAPK/MK2. Cofilin is an actin severing protein and regulates membrane ruffling, lamellipodia formation and cell migration. PIC inhibited cofilin activity by enhancing its phosphorylation at Ser3. PIC activated LIM kinase 1 (LIMK1), an upstream kinase of cofilin in a p38 MAPK-dependent manner. We concluded that the activation of PKR suppressed cell motility by regulating the p38 MAPK/MK2/LIMK/cofilin pathway.

Increased Expression of the dsRNA-Activated Protein Kinase PKR in Breast Cancer Promotes Sensitivity to Doxorubicin

It has been reported that the expression and activity of the interferon-inducible, dsRNA-dependent protein kinase, PKR, is increased in mammary carcinoma cell lines and primary tumor samples. To extend these findings and determine how PKR signaling may affect breast cancer cell sensitivity to chemotherapy, we measured PKR expression by immunohistochemical staining of 538 cases of primary breast cancer and normal tissues. Significantly, PKR expression was elevated in ductal, lobular and squamous cell carcinomas or lymph node metastases but not in either benign tumor specimens or cases of inflammation compared to normal tissues. Furthermore, PKR expression was increased in precancerous stages of mammary cell hyperplasia and dysplasia compared to normal tissues, indicating that PKR expression may be upregulated by the process of tumorigenesis. To test the function of PKR in breast cancer, we generated MCF7, T-47D and MDA-MB-231 breast cancer cell lines with significantly reduced PKR expression by siRNA knockdown. Importantly, while knockdown of PKR expression had no effect on cell proliferation under normal growth conditions, MCF7, T-47D or MDA-MB-231 cells with reduced PKR expression or treated with a small molecule PKR inhibitor were significantly less sensitive to doxorubicin or H2O2-induced toxicity compared to control cells. In addition, the rate of eIF2α phosphorylation following treatment with doxorubicin was delayed in breast cancer cell lines with decreased PKR expression. Significantly, treatment of breast cancer lines with reduced PKR expression with either interferon-α, which increases PKR expression, or salubrinal, which increases eIF2α phosphorylation, restored doxorubicin sensitivity to normal levels. Taken together these results indicate that increased PKR expression in primary breast cancer tissues may serve as a biomarker for response to doxorubicin-containing chemotherapy and that future therapeutic approaches to promote PKR expression/activation and eIF2α phosphorylation may be beneficial for the treatment of breast cancer.

Functional Analysis of Rift Valley Fever Virus NSs Encoding a Partial Truncation

Rift Valley fever virus (RVFV), belongs to genus Phlebovirus of the family Bunyaviridae, causes high rates of abortion and fetal malformation in infected ruminants as well as causing neurological disorders, blindness, or lethal hemorrhagic fever in humans. RVFV is classified as a category A priority pathogen and a select agent in the U.S., and currently there are no therapeutics available for RVF patients. NSs protein, a major virulence factor of RVFV, inhibits host transcription including interferon (IFN)-β mRNA synthesis and promotes degradation of dsRNA-dependent protein kinase (PKR). NSs self-associates at the C-terminus 17 aa., while NSs at aa.210–230 binds to Sin3A-associated protein (SAP30) to inhibit the activation of IFN-β promoter. Thus, we hypothesize that NSs function(s) can be abolished by truncation of specific domains, and co-expression of nonfunctional NSs with intact NSs will result in the attenuation of NSs function by dominant-negative effect. Unexpectedly, we found that RVFV NSs truncated at aa. 6–30, 31–55, 56–80, 81–105, 106–130, 131–155, 156–180, 181–205, 206–230, 231–248 or 249–265 lack functions of IFN–β mRNA synthesis inhibition and degradation of PKR. Truncated NSs were less stable in infected cells, while nuclear localization was inhibited in NSs lacking either of aa.81–105, 106–130, 131–155, 156–180, 181–205, 206–230 or 231–248. Furthermore, none of truncated NSs had exhibited significant dominant-negative functions for NSs-mediated IFN-β suppression or PKR degradation upon co-expression in cells infected with RVFV. We also found that any of truncated NSs except for intact NSs does not interact with RVFV NSs even in the presence of intact C-terminus self-association domain. Our results suggest that conformational integrity of NSs is important for the stability, cellular localization and biological functions of RVFV NSs, and the co-expression of truncated NSs does not exhibit dominant-negative phenotype.

Mechanism of inhibition of lipopolysaccharide-induced interferon-β production by 2-aminopurine

2-Aminopurine (2-AP) is widely used as an inhibitor for double stranded RNA-dependent protein kinase (PKR). Previously, we reported that 2-AP inhibits Toll-like receptor (TLR) ligand-induced nitric oxide production through the prevention of interferon (IFN)-β production. In this study, we investigated the mechanisms for 2-AP inhibition of lipopolysaccharide (LPS)-induced IFN-β production. A reporter gene assay showed that LPS-induced IFN-β promoter, but not nuclear factor (NF)-κB, activation was significantly inhibited by 2-AP. IFN-β promoter activation induced by the overexpression of Toll/interleukin-1 receptor domain-containing adaptor inducing IFN-β (TRIF) was significantly inhibited by 2-AP in a dose-dependent manner, while TRIF- or myeloid differentiation primary response gene 88-dependent NF-κB activation was not inhibited. IFN-β promoter activation induced by expression of the downstream signaling molecules, tumor necrosis factor receptor-associated factor family member-associated NF-κB activator-binding kinase 1, inhibitor of NF-κB kinase i and a constitutively active mutant of interferon regulatory factor (IRF)-3, was also inhibited by 2-AP. Another PKR inhibitor harboring the imidazolo-oxindole structure, however, did not affect TRIF signaling molecules-induced IFN-β promoter activation, suggesting that the inhibition of IFN-β transcription by 2-AP is independent of PKR inhibition. Further, we examined the effect of 2-AP on LPS-induced IRF-3 activation by immunoblotting. While 2-AP did not affect LPS-induced phosphorylation of IRF-3, nuclear translocation of IRF-3 was inhibited. Moreover, we revealed that LPS-induced phosphorylation of Akt, another key molecule involved in IRF-3 activation, was inhibited by 2-AP. These results suggest that 2-AP inhibits nuclear translocation of phosphorylated-IRF-3 by inhibiting Akt activation.

The Dominant-Negative Inhibition of Double-Stranded RNA-Dependent Protein Kinase PKR Increases the Efficacy of Rift Valley Fever Virus MP-12 Vaccine

Rift Valley fever virus (RVFV), belonging to the genus Phlebovirus, family Bunyaviridae, is endemic to sub-Saharan Africa and causes a high rate of abortion in ruminants and hemorrhagic fever, encephalitis, or blindness in humans. MP-12 is the only RVFV strain excluded from the select-agent rule and handled at a biosafety level 2 (BSL2) laboratory. MP-12 encodes a functional major virulence factor, the NSs protein, which contributes to its residual virulence in pregnant ewes. We found that 100% of mice subcutaneously vaccinated with recombinant MP-12 (rMP12)-murine PKRN167 (mPKRN167), which encodes a dominant-negative form of mouse double-stranded RNA (dsRNA)-dependent protein kinase (PKR) in place of NSs, were protected from wild-type (wt) RVFV challenge, while 72% of mice vaccinated with MP-12 were protected after challenge. rMP12-mPKRN167 induced alpha interferon (IFN-α) in sera, accumulated RVFV antigens in dendritic cells at the local draining lymph nodes, and developed high levels of neutralizing antibodies, while parental MP-12 induced neither IFN-α nor viral-antigen accumulation at the draining lymph node yet induced a high level of neutralizing antibodies. The present study suggests that the expression of a dominant-negative PKR increases the immunogenicity and efficacy of live-attenuated RVFV vaccine, which will lead to rational design of safe and highly immunogenic RVFV vaccines for livestock and humans.

Toll-like receptor 3 signaling inhibits double-stranded RNA-induced apoptosis of macrophages (168.21)

Abstract Most viruses produce dsRNA during their infection. Recognition of dsRNA by TLR3, PKR, RIG-I, or MDA-5 initiates innate immune responses, RIG-I and MDA-5 are sensors for dsRNA to induce proinflammatory cytokines or type I interferon, whereas PKR mediates dsRNA-induced apoptosis. Here, we reported that TLR3 deficiency enhances apoptosis of macrophages in response to poly(I:C) as well as Japanese encephalitis virus (JEV). Unexpectedly, TLR3 deficiency markedly increased the phosphorylation of PKR, and inhibition of PKR by RNAi or a PKR-specific inhibitory oligonucleotide (ION) reduced poly(I:C)-induced apoptosis of TLR3-/- macrophages. Stimulation of TLR3 by poly(I:C) activates PI3K and NF-κB signaling pathways, and inhibition of either PI3K or NF-κB enhanced the poly(I:C)-induced apoptosis of macrophages. Further analysis revealed that TLR3 deficiency, as well as inhibition of PI3K or NF-κB, markedly induced p58IPK, a PKR inhibitor, and inhibition of p58IPK by RNAi reduced the apoptosis of poly(I:C)-induced TLR3-/- macrophages. Lastly, TLR3-/- mice are more resistant to JEV infection, but inhibition of apoptosis by caspase inhibitors reduces their resistance to lethal poly(I:C) stimulation or JEV infection. Together, our findings suggest that stimulation of TLR3 by viral dsRNA may activate PI3K and NF-κB, which induces the p58IPK to suppress the phosphorylation of PKR, thus protecting dsRNA-stimulated or virus-infected cells from apoptosis to facilitate the virus propagation.

Abstract 2009: PKR induces apoptosis via PP2A activation and Bcl-2 dephosphorylation in IL-3 dependent murine bone marrow stem/progenitor cells

Abstract The Interferon-induced double-stranded RNA-activated protein kinase (PKR) was originally found to be activated by viral infection leading to inhibition of new protein synthesis and apoptosis. Recently multiple studies have reported decreased expression and function of PKR in leukemia cells, although the exact role of PKR in the process of oncogenesis remains unclear. Since leukemias are clonal disorders originating in a primitive pluripotent leukemic stem cell (LSC) which is regulated by multi-potential hematopoietic growth factors including interleukin-3 (IL-3). Thus, studying how PKR regulates apoptosis in IL-3 dependent (CD123+) pluripotent stem/progenitor cells is expected to reveal PKR's role in leukemia. PKR expression was either knocked down (by 78%) using a specific ShRNA or directly inhibited (57%) by treating cells with a specific PKR inhibitor. Results revealed that inhibition of PKR was associated with a reduction of PP2A activity, a 2.3 fold increase in Bcl-2 phosphorylation and inhibition of apoptosis following treatment with 100uM hydrogen peroxide for 48 hours. In addition, okadaic acid (an inhibitor of PP2A) had a similar effect to PKR inhibition on both Bcl-2 phosphorylation (1.74 fold increase) and stress-induced apoptosis. By contrast, treatment of cells with the PP2A activator, FTY 720, resulted in decreased Bcl-2 phosphorylation (by 2.24 fold) and enhanced apoptosis in response to treatment of cells with chemotherapy (etoposide or doxorubicin) or hydrogen peroxide. To test the role of PKR in growth factor- withdrawal mediated apoptosis in bone marrow stem/progenitor cells, CD123+(IL-3R[[Unsupported Character - Symbol Font ]]+) bone marrow cells from wild type, PKR transgenic (over-expressed) and PKR null(-/-) mice were exposed to different stresses including IL-3 deprivation or hydrogen peroxide. CD123+ cells isolated from PKR null mice had a significantly lower percentage of apoptosis while CD123+ cells from PKR transgenic mice had increased apoptosis compared to cells from wild type littermate mice. These results demonstrate that PKR negatively affects hematopoietic cell survival by enhancing apoptosis following stress in a mechanism that involves PP2A activation and Bcl2 dephosphorylation. Taken together these results may indicate that inhibition of PKR's pro-apoptotic function in bone marrow pluripotent stem/progenitor cells may contribute to tumorigenesis in hematopoietic malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2009. doi:1538-7445.AM2012-2009

PKR plays a positive role in osteoblast differentiation by regulating GSK-3β activity through a β-catenin-independent pathway

Double-stranded RNA-dependent protein kinase (PKR) is involved in various cellular functions. We previously reported that PKR regulates osteoblast differentiation, but the specific mechanisms by which this occurs remain unclear. In this study, we investigated the role of PKR in Glycogen synthase kinase 3β (GSK-3β) regulation of osteoblast differentiation. Lithium chloride (LiCl), a GSK-3β inhibitor, increased GSK-3β phosphorylation in MC3T3-E1 and MG-63 cells. LiCl also inhibited Runx2 and expression of its regulated genes, causing inhibition of Alkaline phosphatase activity and mineralization. LiCl injection to the calvaria in mice suppressed bone formation. Further, GSK-3β phosphorylation was increased in osteoblasts, by Akt-independent mechanisms, in which PKR was constitutively inactivated. A PKR inhibitor, 2-aminopurine, also induced GSK-3β phosphorylation in MC3T3-E1 and MG-63 cells. Further, Runx2 and its regulated genes were inhibited in PKR-inactivated osteoblasts, and differentiation was suppressed through a β-catenin-independent pathway. PKR positively regulates the differentiation of osteoblasts by mediating GSK-3β activity through a β-catenin-independent pathway.