2-methoxyestradiol-induced cell death in osteosarcoma cells is preceded by cell cycle arrest

2-Methoxyestradiol (2-ME), a naturally occurring mammalian metabolite of 17beta-Estradiol (E2), induces cell death in osteosarcoma cells. To further understand the molecular mechanisms of action, we have investigated cell cycle progression in 2-ME-treated human osteosarcoma (MG63, SaOS-2 and LM7 [corrected]) cells. At 5 microM, 2-ME induced growth arrest by inducing a block in cell cycle; 2-ME-treatment resulted in 2-fold increases in G1 phase cells and a decrease in S phase cells in MG63 and SaOS-2 osteosarcoma cell lines, compared to the appropriate vehicle controls. 2-ME-treatment induced a threefold increase in the G2 phase in LM7 [corrected] osteosarcoma cells. The results demonstrated steroid specificity, as the tumorigenic metabolite, 16alpha-hydroxyestradiol (16-OHE), did not have any effect on cell cycle progression in osteosarcoma cells. The cell cycle arrest coincided with an increase in expression of the cell cycle markers p21, p27 and p53 proteins in 2-ME-treated osteosarcoma cells. Also, MG63 cells, transiently transfected with cDNA for a 'loss of function mutant' RNA-dependent protein kinase (PKR) protein, were resistant to 2-ME-induced cell cycle arrest. These results suggest that 2-ME works in concert with factors regulating cell cycle progression, and cell cycle arrest precedes cell death in 2-ME-treated osteosarcoma cells.     

Hypothalamic lipotoxicity and the metabolic syndrome

Ectopic accumulation of lipids in peripheral tissues, such as pancreatic β cells, liver, heart and skeletal muscle, leads to lipotoxicity, a process that contributes substantially to the pathophysiology of insulin resistance, type 2 diabetes, steatotic liver disease and heart failure. Current evidence has demonstrated that hypothalamic sensing of circulating lipids and modulation of hypothalamic endogenous fatty acid and lipid metabolism are two bona fide mechanisms modulating energy homeostasis at the whole body level. Key enzymes, such as AMP-activated protein kinase (AMPK) and fatty acid synthase (FAS), as well as intermediate metabolites, such as malonyl-CoA and long-chain fatty acids-CoA (LCFAs-CoA), play a major role in this neuronal network, integrating peripheral signals with classical neuropeptide-based mechanisms. However, one key question to be addressed is whether impairment of lipid metabolism and accumulation of specific lipid species in the hypothalamus, leading to lipotoxicity, have deleterious effects on hypothalamic neurons. In this review, we summarize what is known about hypothalamic lipid metabolism with focus on the events associated to lipotoxicity, such as endoplasmic reticulum (ER) stress in the hypothalamus. A better understanding of these molecular mechanisms will help to identify new drug targets for the treatment of obesity and metabolic syndrome.     

Double-stranded RNA-dependent protein kinase (PKR) is a stress-responsive kinase that induces NFkappaB-mediated resistance against mercury cytotoxicity

The interferon-inducible, double-stranded (ds)RNA-dependent protein kinase (PKR) plays a major role in antiviral defense mechanisms where it down-regulates translation via phosphorylation of eukaryotic translation initiation factor 2alpha. PKR is also involved in the activation of nuclear factor kappaB (NFkappaB) through activation of the IkappaB kinase complex. Activation of PKR can occur in the absence of dsRNA and in such case is controlled by intracellular regulators like the PKR-activating protein (PACT), the PKR inhibitor p58(IPK), or heat-shock proteins (Hsp). These regulators are activated by stress stimuli, supporting a role for PKR in response to stress; however the final outcome of PKR activation in stress situations is unclear. We present here evidence that expression and activation of PKR contributes to an increased cellular resistance to mercury cytotoxicity. In two cell lines constitutively expressing PKR (THP-1 and Molt-3), treatment with the PKR inhibitor 2-aminopurine increases their sensitivity to mercury. In contrast, Ramos cells, which do not constitutively express PKR, present an increased resistance to mercury when PKR expression is induced by polyIC or interferon-beta treatment. This protective effect is inhibited by 2-aminopurine. We also show that exposure of Ramos cells to mercury leads to the induction of Hsp70. Treatment of cells with Hsp70 or NFkappaB inhibitors suppresses the PKR-dependent protection. We propose a model where PKR, modulated by Hsp70, activates a NFkappaB-mediated protective pathway. Because the cytotoxicity of mercury is primarily due to the generation of reactive oxygen species, our results suggest a more general function of PKR in the mechanisms of cellular response to oxidative stress.     

A protein-kinase, IFN-inducible double-stranded RNA dependent inhibitor and repressor of p58 (PRKRIR) enhances type I IFN-mediated antiviral response through the stability control of RIG-I protein

The cellular RIG-I-like receptor (RLR) senses pathogenic RNA molecular patterns and transmits signals for type I interferon (IFN) production. It acts as a center for antiviral responses, and large numbers of RIG-I (retinoic acid inducible gene-I) interacting proteins are identified as signaling regulators. In the present study, we report PRKRIR, a negative regulator of PKR inhibitor, as a novel RIG-I interacting protein. In HEK293FT cells, PRKRIR synergistically enhances type I IFN production mediated by a signal activated- or constitutively active form of RIG-I. The C-terminal domain of the PRKRIR was required for physical interaction and the signal augmentation. The PRKRIR blocks poly-ubiquitination and protein degradation of RIG-I, thereby increasing cellular levels of RIG-I proteins. Furthermore, overexpression of PRKRIR, along with a signal activated- or constitutively active form of RIG-I, efficiently inhibits virus replication in the infected host. In conclusion, PRKRIR provides a novel positive regulator controlling the RIG-I-IFN production system through protein stability control.     

Sindbis virus replication, is insensitive to rapamycin and torin1, and suppresses Akt/mTOR pathway late during infection in HEK cells

Genetically engineered Sindbis viruses (SIN) are excellent oncolytic agents in preclinical models. Several human cancers have aberrant Akt signaling, and kinase inhibitors including rapamycin are currently tested in combination therapies with oncolytic viruses. Therefore, it was of interest to delineate possible cross-regulation between SIN replication and PI3K/Akt/mTOR signaling. Here, using HEK293T cells as host, we report the following key findings: (a) robust SIN replication occurs in the presence of mTOR specific inhibitors, rapamycin and torin1 or Ly294002 – a PI3K inhibitor, suggesting a lack of requirement for PI3K/Akt/mTOR signaling; (b) suppression of phosphorylation of Akt, mTOR and its effectors S6, and 4E-BP1 occurs late during SIN infection: a viral function that may be beneficial in counteracting cellular drug resistance to kinase inhibitors; (c) Ly294002 and SIN act additively to suppress PI3K/Akt/mTOR pathway with little effect on virus release; and (d) SIN replication induces host translational shut off, phosphorylation of eIF2α and apoptosis. This first report on the potent inhibition of Akt/mTOR signaling by SIN replication, bolsters further studies on the development and evaluation of engineered SIN genotypes in vitro and in vivo for unique cytolytic functions.     

Heparin activates PKR by inducing dimerization

Protein kinase R (PKR) is an interferon-induced kinase that plays a pivotal role in the innate immunity pathway. PKR is activated to undergo autophosphorylation upon binding to double-stranded RNAs or RNAs that contain duplex regions. Activated PKR phosphorylates the α subunit of eukaryotic initiation factor 2, thereby inhibiting protein synthesis. PKR is also activated by heparin, a highly sulfated glycosaminoglycan. We have used biophysical methods to define the mechanism of PKR activation by heparin. Heparins as short as hexasaccharide bind strongly to PKR and activate autophosphorylation. In contrast to double-stranded RNA, heparin activates PKR by binding to the kinase domain. Analytical ultracentrifugation measurements support a thermodynamic linkage model where heparin binding allosterically enhances PKR dimerization, thereby activating the kinase. These results indicate that PKR can be activated by small molecules and represents a viable target for the development of novel antiviral agents.     

MicroRNA-29b Regulates Ethanol-induced Neuronal Apoptosis in the Developing Cerebellum through SP1/RAX/PKR Cascade

Neuronal loss is a prominent etiological factor for fetal alcohol spectrum disorders. The cerebellum is one of the areas in the developing central nervous system that is most sensitive to ethanol, especially during the temporal window of ethanol vulnerability. MicroRNAs are small, non-coding RNAs capable of regulating diverse cellular functions including apoptosis. Ethanol exposure has been shown to interfere with the expression of microRNAs. However, the role of microRNAs in ethanol neurotoxicity is still not clear. In the present study, we identified a particular microRNA, miR-29b, as a novel target of ethanol in the developing cerebellar granule neurons. We discovered that ethanol exposure suppressed miR-29b and induced neuronal apoptosis. Overexpression of miR-29b rendered neurons protection against ethanol-induced apoptosis. Furthermore, our data indicated that miR-29b mediated ethanol neurotoxicity through the SP1/RAX/PKR cascade. More importantly, the expression of miR-29b is developmentally regulated, which may account for, at least partially, the temporal window of ethanol sensitivity in the developing cerebellum.     

Battling for Ribosomes: Translational Control at the Forefront of the Antiviral Response

In the early stages of infection, gaining control of the cellular protein synthesis machinery including its ribosomes is the ultimate combat objective for a virus. To successfully replicate, viruses unequivocally need to usurp and redeploy this machinery for translation of their own mRNA. In response, the host triggers global shutdown of translation while paradoxically allowing swift synthesis of antiviral proteins as a strategy to limit collateral damage. This fundamental conflict at the level of translational control defines the outcome of infection. As part of this special issue on molecular mechanisms of early virus–host cell interactions, we review the current state of knowledge regarding translational control during viral infection with specific emphasis on protein kinase RNA-activated and mammalian target of rapamycin-mediated mechanisms. We also describe recent technological advances that will allow unprecedented insight into how viruses and host cells battle for ribosomes.     

siRNA binding proteins of microglial cells: PKR is an unanticipated ligand

Small interfering RNA (siRNA), double-stranded RNA (dsRNA) 21-23 nucleotides (nt) long with two nt 3' overhangs, has been shown to mediate powerful sequence-specific gene silence in mammalian cells through RNA interference (RNAi). Due to its high efficiency and high specificity siRNA has been used as a powerful post genomic tool and a potent therapeutic candidate. However, there is still a lot to learn about the mobility of siRNA inside cells and the cellular factors that might interfere with the specificity and activity of siRNA. Microglia are the brain's effector cells of the innate immune system and suitable targets in the development of novel therapeutic strategies. Here, we show the cellular uptake and intracellular distribution of siRNA in murine microglial N9 cells. siRNA was internalized by microglial N9 cells without transfection reagent and mainly localized to the endosomes However, no significant gene silencing effects were observed. Its cellular uptake and cellular distribution pattern were similar with that of a same length single stranded DNA (ssDNA). Further, cellular binding proteins of siRNA were purified and identified by mass spectrometry. Negative control siRNA and siRNA targeted to beta-actin were used in this part of experiment. Most of the siRNA binding proteins for negative control siRNA and siRNA targeted to beta-actin were dsRNA-binding proteins, such as dsRNA-dependent protein kinase R (PKR). Furthermore, both control siRNA and siRNA targeted to beta-actin activated PKR in N9 cells, which suggest that siRNA might cause off-target effects through activation of PKR.     

Sweet Killing in Obesity and Diabetes: The Metabolic Role of the BH3-only Protein BIM

Diabetes is a metabolic disorder affecting more than 400 million individuals and their families worldwide. The major forms of diabetes (types 1 and 2) are characterized by pancreatic β-cell dysfunction and, in some cases, loss of β-cell mass causing hyperglycemia due to absolute or relative insulin deficiency. The BCL-2 homology 3 (BH3)-only protein BIM has a wide role in apoptosis induction in cells. In this review, we describe the apoptotic mechanisms mediated by BIM activation in β cells in obesity and both forms of diabetes. We focus on molecular pathways triggered by inflammation, saturated fats, and high levels of glucose. Besides its role in cell death, BIM has been implicated in the regulation of mitochondrial oxidative phosphorylation and cellular metabolism in hepatocytes. BIM is both a key mediator of pancreatic β-cell death and hepatic insulin resistance and is thus a potential therapeutic target for novel anti-diabetogenic drugs. We consider the implications and challenges of targeting BIM in the treatment of the disease.