Curcumin Inhibits Rift Valley Fever Virus Replication in Human Cells

Rift Valley fever virus (RVFV) is an arbovirus that is classified as a select agent, an emerging infectious virus, and an agricultural pathogen. Understanding RVFV-host interactions is imperative to the design of novel therapeutics. Here, we report that an infection by the MP-12 strain of RVFV induces phosphorylation of the p65 component of the NFκB cascade. We demonstrate that phosphorylation of p65 (serine 536) involves phosphorylation of IκBα and occurs through the classical NFκB cascade. A unique, low molecular weight complex of the IKK-β subunit can be observed in MP-12-infected cells, which we have labeled IKK-β2. The IKK-β2 complex retains kinase activity and phosphorylates an IκBα substrate. Inhibition of the IKK complex using inhibitors impairs viral replication, thus alluding to the requirement of an active IKK complex to the viral life cycle. Curcumin strongly down-regulates levels of extracellular infectious virus. Our data demonstrated that curcumin binds to and inhibits kinase activity of the IKK-β2 complex in infected cells. Curcumin partially exerts its inhibitory influence on RVFV replication by interfering with IKK-β2-mediated phosphorylation of the viral protein NSs and by altering the cell cycle of treated cells. Curcumin also demonstrated efficacy against ZH501, the fully virulent version of RVFV. Curcumin treatment down-regulated viral replication in the liver of infected animals. Our data point to the possibility that RVFV infection may result in the generation of novel versions of host components (such as IKK-β2) that, by virtue of altered protein interaction and function, qualify as unique therapeutic targets.     

Chemotactic and inflammatory responses in the liver and brain are associated with pathogenesis of Rift Valley fever virus infection in the mouse

Rift Valley fever virus (RVFV) is a major human and animal pathogen associated with severe disease including hemorrhagic fever or encephalitis. RVFV is endemic to parts of Africa and the Arabian Peninsula, but there is significant concern regarding its introduction into non-endemic regions and the potentially devastating effect to livestock populations with concurrent infections of humans. To date, there is little detailed data directly comparing the host response to infection with wild-type or vaccine strains of RVFV and correlation with viral pathogenesis. Here we characterized clinical and systemic immune responses to infection with wild-type strain ZH501 or IND vaccine strain MP-12 in the C57BL/6 mouse. Animals infected with live-attenuated MP-12 survived productive viral infection with little evidence of clinical disease and minimal cytokine response in evaluated tissues. In contrast, ZH501 infection was lethal, caused depletion of lymphocytes and platelets and elicited a strong, systemic cytokine response which correlated with high virus titers and significant tissue pathology. Lymphopenia and platelet depletion were indicators of disease onset with indications of lymphocyte recovery correlating with increases in G-CSF production. RVFV is hepatotropic and in these studies significant clinical and histological data supported these findings; however, significant evidence of a pro-inflammatory response in the liver was not apparent. Rather, viral infection resulted in a chemokine response indicating infiltration of immunoreactive cells, such as neutrophils, which was supported by histological data. In brains of ZH501 infected mice, a significant chemokine and pro-inflammatory cytokine response was evident, but with little pathology indicating meningoencephalitis. These data suggest that RVFV pathogenesis in mice is associated with a loss of liver function due to liver necrosis and hepatitis yet the long-term course of disease for those that might survive the initial hepatitis is neurologic in nature which is supported by observations of human disease and the BALB/c mouse model.     

Rift Valley fever virus 78kDa envelope protein attenuates virus replication in macrophage-derived cell lines and viral virulence in mice

Rift Valley fever virus (RVFV) is a mosquito-borne bunyavirus with a wide host range including ruminants and humans. RVFV outbreaks have had devastating effects on public health and the livestock industry in African countries. However, there is no approved RVFV vaccine for human use in non-endemic countries and no FDA-approved antiviral drug for RVFV treatment. The RVFV 78kDa protein (P78), which is a membrane glycoprotein, plays a role in virus dissemination in the mosquito host, but its biological role in mammalian hosts remains unknown. We generated an attenuated RVFV MP-12 strain-derived P78-High virus and a virulent ZH501 strain-derived ZH501-P78-High virus, both of which expressed a higher level of P78 and carried higher levels of P78 in the virion compared to their parental viruses. We also generated another MP-12-derived mutant virus (P78-KO virus) that does not express P78. MP-12 and P78-KO virus replicated to similar levels in fibroblast cell lines and Huh7 cells, while P78-High virus replicated better than MP-12 in Vero E6 cells, fibroblast cell lines, and Huh7 cells. Notably, P78-High virus and P78-KO virus replicated less efficiently and more efficiently, respectively, than MP-12 in macrophage cell lines. ZH501-P78-High virus also replicated poorly in macrophage cell lines. Our data further suggest that inefficient binding of P78-High virus to the cells led to inefficient virus internalization, low virus infectivity and reduced virus replication in a macrophage cell line. P78-High virus and P78-KO virus showed lower and higher virulence than MP-12, respectively, in young mice. ZH501-P78-High virus also exhibited lower virulence than ZH501 in mice. These data suggest that high levels of P78 expression attenuate RVFV virulence by preventing efficient virus replication in macrophages. Genetic alteration leading to increased P78 expression may serve as a novel strategy for the attenuation of RVFV virulence and generation of safe RVFV vaccines.     

Signal pathways involved in activation of p70S6K and phosphorylation of 4E-BP1 following exposure of multiple myeloma tumor cells to interleukin-6

Interleukin-6 (IL-6) is a prominent tumor growth factor for malignant multiple myeloma cells. In addition to its known activation of the Janus tyrosine kinase-STAT and RAS-MEK-ERK pathways, recent work suggests that IL-6 can also activate the phosphatidylinositol 3-kinase (PI3-K)/AKT kinase pathway in myeloma cells. Because activation of the PI3-K/AKT as well as RAS-MEK-ERK pathways may result in downstream stimulation of the p70(S6K) (p70) and phosphorylation of the 4E-BP1 translational repressor, we assessed these potential molecular targets in IL-6-treated myeloma cells. IL-6 rapidly activated p70 kinase activity and p70 phosphorylation. Activation was inhibited by wortmannin, rapamycin, and the ERK inhibitors PD98059 and UO126, as well as by a dominant negative mutant of AKT. The concurrent requirements for both ERK and PI3-K/AKT appeared to be a result of their ability to phosphorylate p70 on different residues. In contrast, IL-6-induced phosphorylation of 4E-BP1 was inhibited by rapamycin, wortmannin, and dominant negative AKT but ERK inhibitors had no effect, indicating ERK function was dispensable. In keeping with these data, a dominant active AKT mutant was sufficient to induce 4E-BP1 phosphorylation but could not by itself activate p70 kinase activity. Prevention of IL-6-induced p70 activation and 4E-BP1 phosphorylation by the mammalian target of rapamycin inhibitors rapamycin and CCI-779 resulted in inhibition of IL-6-induced myeloma cell growth. These results indicate that both ERK and PI3-K/AKT pathways are required for optimal IL-6-induced p70 activity, but PI3-K/AKT is sufficient for 4E-BP1 phosphorylation. Both effects are mediated via mammalian target of rapamycin function, and, furthermore, these effects are critical for IL-6-induced tumor cell growth.     

MAPKs and Mst1/Caspase-3 pathways contribute to H2B phosphorylation during UVB-induced apoptosis

Apoptosis is a highly coordinated or programmed cell suicide mechanism in eukaryotes. Histone modification is associated with nuclear events in apoptotic cells. Specifically H2B phosphorylation at serine 14 (Ser14) catalyzed by Mst1 kinase has been linked to chromatin condensation during apoptosis. We report that activation of MAPKs (ERK1/2, JNK1/2 and p38) together with Mst1 and caspase-3 is required for phosphorylation of H2B (Ser14) during ultraviolet B light (UVB)-induced apoptosis. UVB can trigger activation of MAPKs and induce H2B phosphorylation at Ser14 but not acetylation in a time-dependent manner. Inhibition of ERK1/2, JNK1/2 or p38 activity blocked H2B phosphorylation (Ser14). Furthermore, caspase-3 was activated by UVB to regulate Mst1 activity, which phosphorylates H2B at Ser14, leading to chromatin condensation. Full inhibition of caspase-3 activity reduced Mst1 activation and partially inhibited H2B phosphorylation (Ser14), but ERK1/2, JNK1/2 and p38 activities were not affected. Taken together, these data revealed that H2B phosphorylation is regulated by both MAPKs and caspase-3/Mst1 pathways during UVB-induced apoptosis.     

HSP70 deficiency results in activation of c-Jun N-terminal Kinase, extracellular signal-regulated kinase, and caspase-3 in hyperosmolarity-induced apoptosis

In this study we examined the function of heat shock protein 70 (HSP70) in the hyperosmolarity-induced apoptotic pathway using hsp70.1-/-mouse embryonic fibroblasts (MEFs). When the cells were exposed to hyperosmotic stress, an absence of HSP70 negatively affected cell viability. Caspase-9 and caspase-3 were rapidly activated, and extensive cleavage occurred in focal adhesion and cytoskeletal molecules in the hsp70.1-/-MEFs. In contrast, hsp70.1+/+ MEFs exhibited no caspase-9 or caspase-3 activation and finally recovered intact cell morphology when cells were shifted back to an isosmotic state. Because HSP70 might be involved in the regulation of mitogen-activated protein kinase (MAPK) activities with regard to various cellular activities, we also monitored MAPK phosphorylation. The absence of HSP70 affected c-Jun N-terminal kinase phosphorylation. However, it had no effect on p38. Sustained phosphorylation of extracellular signal-regulated kinase (ERK) was observed during the hyperosmolarity-induced apoptosis of hsp70.1-/-MEFs. Inhibition of ERK activity by the treatment of PD98059 accelerated the apoptotic pathway. ERK phosphorylation was precisely correlated with shift of mitogen-activated protein kinase phosphatase-3 from the soluble to insoluble fraction. Our results demonstrate that the inhibitory effect of HSP70 on caspase-3 activation is sufficient to inhibit apoptosis and that HSP70 exhibits regulatory functions to c-Jun N-terminal kinase and ERK phosphorylation in hyperosmolarity-induced apoptosis.     

DPP-4 Inhibitor Attenuates Toxic Effects of Indoxyl Sulfate on Kidney Tubular Cells

Diabetic nephropathy is a common causative factor of chronic kidney disease (CKD). DPP-4 inhibitor has the ability to improve kidney function and renal microvasculature. In the present study, we investigate the deleterious effects of IS on proximal tubular cells and the protective role of DPP-4 inhibitor. Human kidney 2 (HK-2) cells were exposed to IS in the presence or absence of DPP-4 inhibitor. Effects of DPP-4 inhibitor on viability of HK-2 cells were determined by MTT assay. Reactive oxygen species (ROS) production was examined using fluorescent microscopy. Levels of cleaved caspase-3, transforming growth factor-beta (TGF-β), α-smooth muscle actin (α-SMA) and NF-kappaB p65 and phosphorylation of AKT and extracellular signal-regulated kinase (ERK) were detected by immunoblotting. Production of ROS and level of cleaved caspase-3 were increased by IS in a dose-dependent manner. The phosphorylation of AKT and ERK p65 were decreased alongside activation of NF-κB. Expression of TGF-β and α-SMA, were upregulated in IS-treated HK-2 cells. Treatment with DPP-4 inhibitor resulted in a significant increase in cell viability and a decrease of ROS production in IS-treated HK-2 cells. DPP-4 inhibitor restored IS-induced deactivations of AKT and ERK and inhibited activation of NF-κB in IS-treated HK-2 cells. Moreover, DPP-4 inhibitor could also attenuate IS-induced up-regulation of TGF-β and α-SMA expression. These findings suggest that DPP-4 inhibitor possesses anti-apoptotic activity to ameliorate the IS-induced renal damage, which may be partly attributed to regulating ROS/p38MAPK/ERK and PI3K-AKT pathways as well as downstream NF-κB signaling pathway.     

FTY720 alleviates coxsackievirus B3-induced myocarditis and inhibits viral replication through regulating sphingosine 1-phosphate receptors and AKT/caspase-3 pathways

Fingolimod (FTY720) after phosphorylation, as the ligand of sphingosine 1-phosphate receptors (S1PRs), plays an important role in cell proliferation and differentiation. In this article, FTY720 in the treatment of coxsackievirus B3 (CVB3)-induced viral myocarditis was closely related to apoptosis and AKT/caspase-3 apoptotic pathways. We found that CVB3 inhibited myocardial apoptosis at the early stage with upregulating p-AKT level and downregulating activated caspase-3 level for replication of virus progeny, whereas it promoted apoptosis at a late stage with downregulating p-AKT and upregulating activated caspase-3 for releasing the newly synthesized virus to spread. Interestingly, FTY720 could reverse this trend; it promoted apoptosis at an early stage and inhibited apoptosis at the late stage in vivo and vitro, which proved the antiviral effect. We also found that S1PR1, S1PR4, and S1PR5, rather than S1PR2 and S1PR3, were regulated by FTY720 in this process. The results confirmed that FTY720 alleviates CVB3-induced myocarditis and inhibits viral replication through regulating S1PRs and AKT/caspase-3 pathways with a bidirectional regulation of apoptosis.     

Amylin-induced cytotoxicity is associated with activation of caspase-3 and MAP kinases

Nanomolar concentrations of human amylin promote death of RINm5F cells in a time- and concentrationdependent manner. Morphological changes of chromatin integrity suggest that cells are predominantly undergoing apoptosis. Human amylin induces significant activation of caspase-3 and strong and sustained phosphorylation of stress-activated protein kinases, c-Jun N-terminal kinase (JNK) and p38, that precedes cell death. Extracellular signal-regulated kinase (ERK) activation was not concomitant with JNK and/or p38 activation. Activation of caspase-3 and mitogen-activated protein kinases (MAPKs) was detected by Western blot analysis. Addition of the MEK1 inhibitor PD 98059 had no effect on amylin-induced apoptosis, suggesting that ERK activation does not play a role in this apoptotic scenario. A correlative inhibition of JNK activation by the immunosuppressive drug FK506, as well as a selective inhibition of p38 MAPK activation by SB 203580, significantly suppressed procaspase-3 processing and the extent of amylin-induced cell death. Moreover, simultaneous pretreatment with both FK506 and SB 203580, or with the caspase-3 inhibitor Ac-DEVD-CHO alone, almost completely abolished procaspase-3 processing and cell death. Thus, our results suggest that amylin-induced apoptosis proceeds through sustained activation of JNK and p38 MAPK followed by caspase-3 activation.     

Suppression of extracellular signal-related kinase and activation of p38 MAPK are two critical events leading to caspase-8- and mitochondria-mediated cell death in phytosphingosine-treated human cancer cells

We previously demonstrated that the phytosphingosine-induced apoptosis was accompanied by the concomitant induction of both the caspase-8-mediated and mitochondrial activation-mediated apoptosis pathways. In the present study, we investigated the role of mitogen-activated protein kinases (MAPKs) in the activation of these two distinct cell death pathways induced by phytosphingosine in human cancer cells. Phytosphingosine caused strong induction of caspase-8 activity and caspase-independent Bax translocation to the mitochondria. A rapid decrease of phosphorylated ERK1/2 and a marked increase of p38 MAPK phosphorylation were observed within 10 min after phytosphingosine treatment. Activation of ERK1/2 by pretreatment with phorbol 12-myristate 13-acetate or forced expression of ERK1/2 attenuated phytosphingosine-induced caspase-8 activation. However, Bax translocation and caspase-9 activation was unaffected, indicating that down-regulation of the ERK activity is specifically required for the phytosphingosine-induced caspase-8-dependent cell death pathway. On the other hand, treatment with SB203580, a p38 MAPK-specific inhibitor, or expression of a dominant negative form of p38 MAPK suppressed phytosphingosine-induced translocation of the proapoptotic protein, Bax, from the cytosol to mitochondria, cytochrome c release, and subsequent caspase-9 activation but did not affect caspase-8 activation, indicating that activation of p38 MAPK is involved in the mitochondrial activation-mediated cell death pathway. Our results suggest that phytosphingosine can utilize two different MAPK signaling pathways for amplifying the apoptosis cascade, enhancing the understanding of the molecular mechanisms utilized by naturally occurring metabolites to regulate cell death. Molecular dissection of the signaling pathways that activate the apoptotic cell death machinery is critical for both our understanding of cell death events and development of cancer therapeutic agents.     

Epigallocatechin-3-gallate inhibits epidermal growth factor receptor signaling pathway. Evidence for direct inhibition of ERK1/2 and AKT kinases

Epidermal growth factor receptor (EGFR) activation is absolutely required for cervical cell proliferation. This suggests that EGFR-inhibitory agents may be of therapeutic value. In the present study, we investigated the effects of epigallocatechin-3-gallate (EGCG), a bioactive green tea polyphenol, on EGFR signaling in cervical cells. EGCG inhibits epidermal growth factor-dependent activation of EGFR, and EGFR-dependent activation of the mitogen-activated protein kinases ERK1/2. EGCG also inhibits EGFR-dependent AKT activity. The EGCG-dependent reduction in ERK and AKT activity is associated with reduced phosphorylation of downstream substrates, including p90RSK, FKHR, and BAD. These changes are associated with increased p53, p21(WAF-1), and p27(KIP-1) levels, reduced cyclin E level, and reduced CDK2 kinase activity. Consistent with these findings, flow cytometry and TUNEL (terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling) staining revealed EGCG-dependent G(1) arrest. Moreover, sustained EGCG treatment caused apoptotic cell death. In addition to inhibiting EGFR, cell-free studies demonstrated that EGCG directly inhibits ERK1/2 and AKT, suggesting that EGCG acts simultaneously at multiple levels to inhibit EGF-dependent signaling. Importantly, the EGCG inhibition is selective, as EGCG does not effect the EGFR-dependent activation of JNK. These results suggest that EGCG acts to selectively inhibit multiple EGF-dependent kinases to inhibit cell proliferation.     

Plasmalogens Rescue Neuronal Cell Death through an Activation of AKT and ERK Survival Signaling

Neuronal cells are susceptible to many stresses, which will cause the apoptosis and neurodegenerative diseases. The precise molecular mechanism behind the neuronal protection against these apoptotic stimuli is necessary for drug discovery. In the present study, we have found that plasmalogens (Pls), which are glycerophospholipids containing vinyl ether linkage at sn-1 position, can protect the neuronal cell death upon serum deprivation. Interestingly, caspse-9, but not caspase-8 and caspase-12, was cleaved upon the serum starvation in Neuro-2A cells. Pls treatments effectively reduced the activation of caspase-9. Furthermore, cellular signaling experiments showed that Pls enhanced phosphorylation of the phosphoinositide 3-kinase (PI3K)-dependent serine/threonine-specific protein kinase AKT and extracellular-signal-regulated kinases ERK1/2. PI3K/AKT inhibitor LY294002 and MAPK/ERK kinase (MEK) inhibitor U0126 treatments study clearly indicated that Pls-mediated cell survival was dependent on the activation of these kinases. In addition, Pls also inhibited primary mouse hippocampal neuronal cell death induced by nutrient deprivation, which was associated with the inhibition of caspase-9 and caspase-3 cleavages. It was reported that Pls content decreased in the brain of the Alzheimer’s patients, which indicated that the reduction of Pls content could endanger neurons. The present findings, taken together, suggest that Pls have an anti-apoptotic action in the brain. Further studies on precise mechanisms of Pls-mediated protection against cell death may lead us to establish a novel therapeutic approach to cure neurodegenerative disorders.     

Genetic Subpopulations of Rift Valley Fever Virus Strains ZH548 and MP-12 and Recombinant MP-12 Strains

Rift Valley fever virus strain MP-12 was generated by serial plaque passages of parental strain ZH548 12 times in MRC-5 cells in the presence of a chemical mutagen, 5-fluorouracil. As a result, MP-12 encoded 4, 9, and 10 mutations in the S, M, and L segments, respectively. Among them, mutations in the M and L segments were responsible for attenuation, while the MP-12 S segment still encoded a virulent phenotype. We performed high-throughput sequencing of MP-12 vaccine, ZH548, and recombinant MP-12 (rMP-12) viruses. We found that rMP-12 contains very low numbers of viral subpopulations, while MP-12 and ZH548 contain 2 to 4 times more viral genetic subpopulations than rMP-12. MP-12 genetic subpopulations did not encode the ZH548 sequence at the 23 MP-12 consensus mutations. On the other hand, 4 and 2 mutations in M and L segments of MP-12 were found in ZH548 subpopulations. Thus, those 6 mutations were no longer MP-12-specific mutations. ZH548 encoded several unique mutations compared to other Egyptian strains, i.e., strains ZH501, ZH1776, and ZS6365. ZH548 subpopulations shared nucleotides at the mutation site common with those in the Egyptian strains, while MP-12 subpopulations did not share those nucleotides. Thus, MP-12 retains unique genetic subpopulations and has no evidence of reversion to the ZH548 sequence in the subpopulations. This study provides the first information regarding the genetic subpopulations of RVFV and shows the genetic stability of the MP-12 vaccine manufactured in MRC-5 cells.     

Apigenin-induced apoptosis is mediated by reactive oxygen species and activation of ERK1/2 in rheumatoid fibroblast-like synoviocytes

Fibroblast-like synovial cells play a crucial role in the pathophysiology of rheumatoid arthritis (RA), as these cells are involved in inflammation and joint destruction. Apigenin, a dietary plant-flavonoid, is known to have many functions in animal cells including anti-proliferative and anticancer activities, but its role in human rheumatoid arthritis fibroblast-like synoviocytes (RA-FLSs) has not been reported. In this study, we investigated the roles of apigenin in RA-FLSs. The survival rate decreased, and apoptotic cell death was induced by apigenin treatment in RA-FLSs. Apigenin treatment resulted in activation of the mitogen-activated protein kinase (MAPK) ERK1/2, and pretreatment with an ERK inhibitor PD98059 dramatically reduced apigenin-induced apoptosis. We found that apigenin-mediated production of a large amount of intracellular reactive oxygen species (ROS) caused activation of ERK1/2 and apoptosis; treatment with the antioxidant Tiron strongly inhibited the apigenin-induced generation of ROS, phosphorylation of ERK1/2, and apoptotic cell death. Apigenin-induced apoptotic cell death was mediated through activation of the effectors caspase-3 and caspase-7, and was blocked by pretreatment with Z-VAD-FMK (a pan-caspase inhibitor). These results showed that apigenin-induced ROS and oxidative stress-activated ERK1/2 caused apoptotic cell death in apigenin-treated RA-FLSs.     

Development of a Novel,Single-Cycle Replicable Rift Valley Fever Vaccine

Rift Valley fever virus (RVFV) (genus Phlebovirus, family Bunyaviridae) is an arbovirus that causes severe disease in humans and livestock in sub-Saharan African countries. Although the MP-12 strain of RVFV is a live attenuated vaccine candidate, neuroinvasiveness and neurovirulence of MP-12 in mice may be a concern when vaccinating certain individuals, especially those that are immunocompromised. We have developed a novel, single-cycle replicable MP-12 (scMP-12), which carries an L RNA, M RNA mutant encoding a mutant envelope protein lacking an endoplasmic reticulum retrieval signal and defective for membrane fusion function, and S RNA encoding N protein and green fluorescent protein. The scMP-12 underwent efficient amplification, then formed plaques and retained the introduced mutation after serial passages in a cell line stably expressing viral envelope proteins. However, inoculation of the scMP-12 into naïve cells resulted in a single round of viral replication, and production of low levels of noninfectious virus-like particles. Intracranial inoculation of scMP-12 into suckling mice did not cause clinical signs or death, a finding which demonstrated that the scMP-12 lacked neurovirulence. Mice immunized with a single dose of scMP-12 produced neutralizing antibodies, whose titers were higher than in mice immunized with replicon particles carrying L RNA and S RNA encoding N protein and green fluorescent protein. Moreover, 90% of the scMP-12-immunized mice were protected from wild-type RVFV challenge by efficiently suppressing viremia and replication of the challenge virus in the liver and the spleen. These data demonstrated that scMP-12 is a safe and immunogenic RVFV vaccine candidate.