Phosphorylation-dependent ubiquitination and degradation of the IFNAR1 chain of Type I interferon (IFN) receptor is regulated by two different pathways one of which is ligand-independent. We report that this pathway is activated by inducers of the endoplasmic reticulum (ER) stress, including viral infection, in a PERK-dependent manner. Upon infection, activation of this pathway promotes phosphorylation-dependent ubiquitination and degradation of IFNAR1, and specifically inhibits Type I IFN signaling and antiviral defenses. Either knock-in of an IFNAR1 mutant insensitive to virus-induced turnover or conditional knockout of PERK prevented ER stress- and virus-induced IFNAR1 degradation while restoring cellular responses to Type I IFN and resistance to viruses. The role of this novel mechanism in pathogenesis of viral infections and therapeutic approaches to their treatment is discussed.

BackgroundThe antiparasitic agent niclosamide has been demonstrated to inhibit the arthropod-borne Zika virus. Here, we investigated the antiviral capacity of niclosamide against dengue virus (DENV) serotype 2 infection in vitro and in vivo.Principle findingNiclosamide effectively retarded DENV-induced infection in vitro in human adenocarcinoma cells (A549), mouse neuroblastoma cells (Neuro-2a), and baby hamster kidney fibroblasts (BHK-21). Treatment with niclosamide did not retard the endocytosis of DENV while niclosamide was unable to enhance the antiviral type I interferon response. Furthermore, niclosamide did not cause a direct effect on viral replicon-based expression. Niclosamide has been reported to competitively inhibit the mTOR (mammalian target of rapamycin), STAT3 (signal transducer and activator of transcription 3), and NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) signaling pathways; however, selective inhibitors of those pathways did not reduce DENV infection. Similar to the vacuolar-type H+-ATPase inhibitor bafilomycin A1, both niclosamide and other protonophores, such as CCCP (carbonyl cyanide m-chlorophenyl hydrazone), and FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone), effectively reduced endosomal acidification and viral dsRNA replication. Co-administration of a single dose of niclosamide partially decreased viral replication, viral encephalitis, and mortality in DENV-infected ICR suckling mice.SignificanceThese results demonstrate that niclosamide diminishes viral infection by hindering endosomal acidification.

Phosphorylation-dependent ubiquitination and ensuing down-regulation and lysosomal degradation of the interferon ␣/␤ receptor chain 1 (IFNAR1) of the receptor for Type I interferons play important roles in limiting the cellular responses to these cytokines. These events could be stimulated either by the ligands (in a Janus kinase-dependent manner) or by unfolded protein response (UPR) inducers including viral infection (in a manner dependent on the activity of pancreatic endoplasmic reticulum kinase). Both ligand-dependent and -independent pathways converge on phosphorylation of Ser 535 within the IFNAR1 degron leading to recruitment of ␤-Trcp E3 ubiquitin ligase and concomitant ubiquitination and degradation. Casein kinase 1␣ (CK1␣) was shown to directly phosphorylate Ser 535 within the ligand-independent pathway. Yet given the constitutive activity of CK1␣, it remained unclear how this pathway is stimulated by UPR. Here we report that induction of UPR promotes the phosphorylation of a proximal residue, Ser 532 , in a pancreatic endoplasmic reticulum kinase-dependent manner. This serine serves as a priming site that promotes subsequent phosphorylation of IFNAR1 within its degron by CK1␣. These events play an important role in regulating ubiquitination and degradation of IFNAR1 as well as the extent of Type I interferon signaling.Ligand-induced down-regulation of cell surface receptors represents a major mode of actions for the branch of signaling that leads to its elimination (1). For example, Type I interferons (including IFN␣ and IFN␤), 2 the cytokines that play a paramount role in anti-viral defense (2) and elicit potent antiproliferative effects (3), stimulate down-regulation of the cell surface levels of their receptor (4, 5). This receptor consists of IFNAR1 and IFNAR2 chains and functions via activation of associated Janus kinase (Tyk2 and Jak1) leading to activating tyrosine phosphorylation of the signal transducers and activators of transcription proteins (STAT1 and STAT2). In turn, these STAT proteins govern transcription of IFN-stimulated genes whose products mediate anti-viral, anti-proliferative, and immunomodulatory functions (reviewed in Refs. 6 and 7). Early studies have reported that IFNAR1 is rapidly down-regulated and degraded upon internalization in response to IFN␣ (8,9).Mechanisms of ligand-induced degradation of IFNAR1 rely on IFN␣/␤-stimulated and Tyk2 catalytic activity-dependent phosphorylation of this receptor chain on Ser 535/539 within a specific phospho-degron (10 -12). This phosphorylation enables the recognition of IFNAR1 by the ␤-Trcp2/HOS F-box protein, followed by the recruitment of the SCF ␤-Trcp E3 ubiquitin ligase (10, 11). This ligase facilitates polyubiquitination of IFNAR1 on a specific cluster of lysines. Through a yet to be identified mechanism, this site-specific ubiquitination results in an exposure of a previously masked linear endocytic motif that enables the recruitment of the AP2 complex and ensuing internalization of IFNAR1 and of entire Type I IFN receptor (13,...

Osmotic stress activates MAPKs, including JNK and p38, which play important roles in cellular stress responses. Transforming growth factor-␤-activated kinase 1 (TAK1) is a member of the MAPK kinase kinase (MAPKKK) family and can activate JNK and p38. TAK1 can also activate IB kinase (IKK) that leads to degradation of IB and subsequent NF-B activation. We found that TAK1 is essential for osmotic stress-induced activation of JNK but is not an exclusive mediator of p38 activation. Furthermore, we found that although TAK1 was highly activated upon osmotic stress, it could not induce degradation of IB or activation of NF-B. These results suggest that TAK1 activity is somehow modulated to function specifically in osmotic stress signaling, leading to the activation of JNK but not of IKK. To elucidate the mechanism underlying this modulation, we screened for potential TAK1-binding proteins. We found that TAO2 (thousand-and-one amino acid kinase 2) associates with TAK1 and can inhibit TAK1-mediated activation of NF-B but not of JNK. We observed that TAO2 can interfere with the interaction between TAK1 and IKK and thus may regulate TAK1 function. TAK1 is activated by many distinct stimuli, including cytokines and stresses, and regulation by TAO2 may be important to activate specific intracellular signaling pathways that are unique to osmotic stress.

We have used a perfusion bellows cell culture system to investigate resveratrolinduced anti-proliferation/apoptosis in a human estrogen receptor (ER)-negative breast cancer cell line (MDA-MB-231). Using an injection system to perfuse media with stilbene, we showed resveratrol (0.5 – 100 μM) to decrease cell proliferation in a concentration-dependent manner. Comparison of influx and medium efflux resveratrol concentrations revealed rapid disappearance of the stilbene, consistent with cell uptake and metabolism of the agent reported by others. Exposure of cells to 10 μM resveratrol for 4 h daily × 6 d inhibited cell proliferation by more than 60%. Variable extracellular acid-alkaline conditions (pH 6.8 – 8.6) affected basal cell proliferation rate, but did not alter anti-proliferation induced by resveratrol. Resveratrol-induced gene expression, including transcription of the most up-regulated genes and pro-apoptotic p53-dependent genes, was not affected by culture pH changes. The microarray findings in the context of induction of anti-proliferation with brief daily exposure of cells to resveratrol—and rapid disappearance of the compound in the perfusion system—are consistent with existence of an accessible initiation site for resveratrol actions on tumor cells, e.g., the cell surface receptor for resveratrol described on integrin αvβ3.

Dihydrotestosterone (DHT) has been shown to promote breast cancer growth via different mechanisms. In addition to binding to ERα, the DHT membrane receptor exists on integrin αvβ3. Resveratrol induces p53-dependent apoptosis via plasma membrane integrin αvβ3. Resveratrol and DHT signals are both transduced by activated ERK1/2; however, DHT promotes cell proliferation in cancer cells, whereas resveratrol is proapoptotic. In this study, we examined the mechanism by which DHT inhibits resveratrolinduced apoptosis in human ERα positive (MCF-7) and negative (MDA-MB-231) breast cancer cells. DHT inhibited resveratrol-stimulated phosphorylation of Ser-15 of p53 in a concentration-dependent manner. These effects of DHT on resveratrol action were blocked by an ERα antagonist, ICI 182,780, in MCF-7 breast cancer cells. DHT inhibited resveratrol-induced nuclear complex of p53-COX-2 formation which is required p53-dependent apoptosis. ChIP studies of COX-2/p53 binding to DNA and expression of p53-responsive genes indicated that DHT inhibited resveratrol-induced p53-directed transcriptional activity. In addition, DHT did inhibit resveratrol-induced COX-2/p53-dependent gene expression. These results suggest that DHT inhibits p53-dependent apoptosis in breast cancer cells by interfering with nuclear COX-2 accumulation which is essential for stimulation of apoptotic pathways. Thus, the surface receptor sites for resveratrol and DHT are discrete and activate ERK1/2-dependent downstream effects on apoptosis that are distinctive. These studies provide new insights into the antagonizing effects of resveratrol versus DHT, an important step toward better understanding and eventually treating breast cancer. It also indicates the complex pathways by which apoptosis is induced by resveratrol in DHT-depleted and -repleted environments.Oncotarget 35867 www.impactjournals.com/oncotarget

An ability to sense pathogens by a number of specialized cell types including the dendritic cells plays a central role in host's defenses. Activation of these cells through the stimulation of the pathogen-recognition receptors induces the production of a number of cytokines including Type I interferons (IFNs) that mediate the diverse mechanisms of innate immunity. Type I IFNs interact with the Type I IFN receptor, composed of IFNAR1 and IFNAR2 chains, to mount the host defense responses. However, at the same time, Type I IFNs elicit potent anti-proliferative and pro-apoptotic effects that could be detrimental for IFN-producing cells. Here, we report that the activation of p38 kinase in response to pathogen-recognition receptors stimulation results in a series of phosphorylation events within the IFNAR1 chain of the Type I IFN receptor. This phosphorylation promotes IFNAR1 ubiquitination and accelerates the proteolytic turnover of this receptor leading to an attenuation of Type I IFN signaling and the protection of activated dendritic cells from the cytotoxic effects of autocrine or paracrine Type I IFN. In this paper we discuss a potential role of this mechanism in regulating the processes of innate immunity.

Ubiquitination of signaling cell surface receptors is a key mechanism regulating the availability of these receptors to interact with extracellular ligands. Accordingly, this regulation determines the sensitivity of cells to the humoral and locally secreted regulators of cell function, proliferation and viability. Alterations in receptor ubiquitination and degradation are often encountered in cancers. Malignant cells utilize modified ubiquitination of signaling receptors to augment or attenuate signaling pathways on the basis of whether the outcome of this signaling is conducive or not for tumor growth and survival. These mechanisms as well as their significance for treatment of human cancers are discussed.