Ultraviolet light inhibits translation through activation of the unfolded protein response kinase PERK in the lumen of the endoplasmic reticulum

Exposure to ultraviolet light can cause inflammation, premature skin aging, and cancer. UV irradiation alters the expression of multiple genes that encode functions to repair DNA damage, arrest cell growth, and induce apoptosis. In addition, UV irradiation inhibits protein synthesis, although the mechanism is not known. In this report, we show that UV irradiation induces phosphorylation of eukaryotic translation initiation factor 2 on the alpha-subunit (eIF2alpha) and inhibits protein synthesis in a dosage- and time-dependent manner. The UV-induced phosphorylation of eIF2alpha was prevented by the overexpression of a non-phosphorylatable mutant of eIF2alpha (S51A). PERK is an eIF2alpha protein kinase localized to the endoplasmic reticulum that is activated by the accumulation of unfolded proteins in the endoplasmic reticulum. Expression of trans-dominant-negative mutants of PERK also prevented eIF2alpha phosphorylation upon UV treatment and protected from the associated translation attenuation. The luminal domain of dominant-negative mutant PERK formed heterodimers with endogenous PERK to inhibit the PERK signaling pathway. In contrast, eIF2alpha phosphorylation was not inhibited by overexpression of a trans-dominant-negative mutant kinase, PKR, supporting the theory that UV-induced eIF2alpha phosphorylation is specifically mediated by PERK. These results support a novel mechanism by which UV irradiation regulates translation via an endoplasmic reticulum-stress signaling pathway.     

Aminosalicylic acid inhibits IkappaB kinase alpha phosphorylation of IkappaBalpha in mouse intestinal epithelial cells

Tumor necrosis factor alpha (TNFalpha)-stimulated nuclear factor (NF) kappaB activation plays a key role in the pathogenesis of inflammatory bowel disease (IBD). Phosphorylation of NFkappaB inhibitory protein (IkappaB) leading to its degradation and NFkappaB activation, is regulated by the multimeric IkappaB kinase complex, including IKKalpha and IKKbeta. We recently reported that 5-aminosalicylic acid (5-ASA) inhibits TNFalpha-regulated IkappaB degradation and NFkappaB activation. To determine the mechanism of 5-ASA inhibition of IkappaB degradation, we studied young adult mouse colon (YAMC) cells by immunodetection and in vitro kinase assays. We show 5-ASA inhibits TNFalpha-stimulated phosphorylation of IkappaBalpha in intact YAMC cells. Phosphorylation of a glutathione S-transferase-IkappaBalpha fusion protein by cellular extracts or immunoprecipitated IKKalpha isolated from cells treated with TNFalpha is inhibited by 5-ASA. Recombinant IKKalpha and IKKbeta autophosphorylation and their phosphorylation of glutathione S-transferase-IkappaBalpha are inhibited by 5-ASA. However, IKKalpha serine phosphorylation by its upstream kinase in either intact cells or cellular extracts is not blocked by 5-ASA. Surprisingly, immunodepletion of cellular extracts suggests IKKalpha is predominantly responsible for IkappaBalpha phosphorylation in intestinal epithelial cells. In summary, 5-ASA inhibits TNFalpha-stimulated IKKalpha kinase activity toward IkappaBalpha in intestinal epithelial cells. These findings suggest a novel role for 5-ASA in the management of IBD by disrupting TNFalpha activation of NFkappaB.     

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.     

Inhibition of mammalian translation initiation by volatile anesthetics

Volatile anesthetics are essential for modern medical practice, but sites and mechanisms of action for any of their numerous cellular effects remain largely unknown. Previous studies with yeast showed that volatile anesthetics induce nutrient-dependent inhibition of growth through mechanisms involving inhibition of mRNA translation. Studies herein show that the volatile anesthetic halothane inhibits protein synthesis in perfused rat liver at doses ranging from 2 to 6%. A marked disaggregation of polysomes occurs, indicating that inhibition of translation initiation plays a key role. Dose- and time-dependent alterations that decrease the function of a variety of translation initiation processes are observed. At 6% halothane, a rapid and persistent increase in phosphorylation of the alpha-subunit of eukaryotic translation initiation factor (eIF)2 occurs. This is accompanied by inhibition of activity of the guanine nucleotide exchange factor eIF2B that is responsible for GDP-GTP exchange on eIF2. At lower doses, neither eIF2alpha phosphorylation nor eIF2B activity is altered. After extended exposure to 6% halothane, alterations in two separate responses regulated by the target of rapamycin pathway occur: 1) redistribution of eIF4E from its translation-stimulatory association with eIF4G to its translation-inactive complex with eIF4E-binding protein-1; and 2) decreased phosphorylation of ribosomal protein S6 (rpS6) with a corresponding decrease in active forms of a kinase that phosphorylates rpS6 (p70(S6K1)). Changes in the association of eIF4E and eIF4G are observed only after extended exposure to low anesthetic doses. Thus dose- and time-dependent alterations in multiple processes permit liver cells to adapt translation to variable degrees and duration of stress imposed by anesthetic exposure.     

TNF inhibited the apoptosis by activation of Akt serine/threonine kinase in the human head and neck squamous cell carcinoma

Tumour necrosis factor (TNF) is known to induce apoptosis, but recently, TNF was shown to promote cell survival, a process regulated by phosphatidylinositol-3-OH kinase (PI3K) and the NFkappaB pathway. In this study, we investigated the relationship between the molecules implicated in regulating TNF-induced cell survival and apoptosis induced by TNF in a human head and neck squamous cell carcinoma cell line (SAS), with special reference to the Akt pathway, one of the pathways related to cell survival. In SAS cells, TNF induced the phosphorylation of Akt at both Ser473 and Thr308, causing the activation of Akt, and also induced the phosphorylation and degradation of IkappaB (inhibitor of NFkappaB). This phosphorylation and degradation was inhibited by pretreating the cells with the PI3K inhibitors, wortmannin or LY294002. The apoptosis of SAS cells induced by TNF was dependent on the concentration: a high concentration of TNF, but not a low concentration, induced apoptosis within 30 h. However, a low concentration of TNF in the presence of wortmannin or LY294002 induced apoptosis. Furthermore, expression of the kinase-negative form of Akt, IKKalpha or IKKbeta, and the undegradable mutant of IkappaB, also induced apoptosis at low concentrations of TNF. When the SAS cells expressed constitutively activated Akt, apoptosis was not induced, even by high concentrations of TNF. These observations suggest that, in the SAS cell line, the PI3K-NFkappaB pathway contributes to TNF-induced cell survival and that inhibition of this pathway accelerates apoptosis.     

Ultraviolet radiation-induced interleukin 6 release in HeLa cells is mediated via membrane events in a DNA damage-independent way

Evidence exists that ultraviolet radiation (UV) affects molecular targets in the nucleus or at the cell membrane. UV-induced apoptosis was found to be mediated via DNA damage and activation of death receptors, suggesting that nuclear and membrane effects are not mutually exclusive. To determine whether participation of nuclear and membrane components is also essential for other UV responses, we studied the induction of interleukin-6 (IL-6) by UV. Exposing HeLa cells to UV at 4 degrees C, which inhibits activation of surface receptors, almost completely prevented IL-6 release. Enhanced repair of UV-mediated DNA damage by addition of the DNA repair enzyme photolyase did not affect UV-induced IL-6 production, suggesting that in this case membrane events predominant over nuclear effects. UV-induced IL-6 release is mediated via NFkappaB since the NFkappaB inhibitor MG132 or transfection of cells with a super-repressor form of the NFkappaB inhibitor IkappaB reduced IL-6 release. Transfection with a dominant negative mutant of the signaling protein TRAF-2 reduced IL-6 release upon exposure to UV, indicating that UV-induced IL-6 release is mediated by activation of the tumor necrosis factor receptor-1. These data demonstrate that UV can exert biological effects mainly by affecting cell surface receptors and that this is independent of its ability to induce nuclear DNA damage.     

Efficient replication by herpes simplex virus type 1 involves activation of the IkappaB kinase-IkappaB-p65 pathway

Infection by herpes simplex virus type 1 (HSV-1) induces a persistent nuclear translocation of NFkappaB. To identify upstream effectors of NFkappaB and their effect on virus replication, we employed mouse embryo fibroblast (MEF)-derived cell lines with deletions of either IKK1 or IKK2, the catalytic subunits of the IkappaB kinase (IKK) complex. Infected MEFs were assayed for virus yield, loss of IkappaBalpha, nuclear translocation of p65, and NFkappaB DNA-binding activity. Absence of either IKK1 or IKK2 resulted in an 86 to 94% loss of virus yield compared to that of normal MEFs, little or no loss of IkappaBalpha, and greatly reduced NFkappaB nuclear translocation. Consistent with reduced virus yield, accumulation of the late proteins VP16 and gC was severely depressed. Infection of normal MEFs, Hep2, or A549 cells with an adenovirus vector expressing a dominant-negative (DN) IkappaBalpha, followed by superinfection with HSV, resulted in a 98% drop in virus yield. These results indicate that the IKK-IkappaB-p65 pathway activates NFkappaB after virus infection. Analysis of NFkappaB activation and virus replication in control and double-stranded RNA-activated protein kinase-null MEFs indicated that this kinase plays no role in the NFkappaB activation pathway. Finally, in cells where NFkappaB was blocked because of DNIkappaB expression, HSV failed to suppress two markers of apoptosis, cell surface Annexin V staining and PARP cleavage. These results support a model in which activation of NFkappaB promotes efficient replication by HSV, at least in part by suppressing a host innate response to virus infection.     

Glucosamine-induced phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 is mediated by the protein kinase R-like endoplasmic-reticulum associated kinase

In diabetic animals, enhanced production of vascular endothelial growth factor is thought to be a major contributor to the development of diabetic retinopathy. In the present study, glucosamine-treated R28 retinal neuronal cells were used as an experimental model system to explore the possible involvement of the hexosamine biosynthetic pathway in the diabetes-induced changes in mRNA translation. Glucosamine treatment enhanced vascular endothelial growth factor production subsequent to changes in phosphorylation of the alpha-subunit of eukaryotic initiation factor 2, with no change in vascular endothelial growth factor mRNA content. Possible mechanisms through which glucosamine might act to increase eukaryotic initiation factor 2alpha phosphorylation include enhanced O-linked glycosylation of protein kinase or phosphatase regulatory proteins and/or induction of oxidative stress. However, increasing global protein O-glycosylation through inhibition of O-beta-N-acetylglucosaminidase did not mimic the effect of glucosamine on eukaryotic initiation factor 2alpha phosphorylation. Likewise, attenuating glucosamine-induced oxidative stress with two different antioxidants did not reduce glucosamine-induced eukaryotic initiation factor 2alpha phosphorylation. Glucosamine treatment was also found to promote eukaryotic initiation factor 2alpha phosphorylation in wild-type mouse embryonic fibroblasts, but not in mouse embryonic fibroblasts lacking the eukaryotic initiation factor 2alpha kinase referred to as RNA-dependent protein kinase-like endoplasmic-reticulum associated kinase, implicating the kinase in the glucosamine-induced increase in eukaryotic initiation factor 2alpha phosphorylation. Overall, the results are consistent with glucosamine causing activation of RNA-dependent protein kinase-like endoplasmic-reticulum associated kinase, which phosphorylates eukaryotic initiation factor 2alpha and consequently upregulates translation of mRNAs encoding specific proteins, such as vascular endothelial growth factor.     

Interleukin-1 (IL-1)-induced TAK1-dependent Versus MEKK3-dependent NFkappaB activation pathways bifurcate at IL-1 receptor-associated kinase modification

Interleukin-1 (IL-1) receptor-associated kinase (IRAK) is phosphorylated after it is recruited to the receptor, subsequently ubiquitinated, and eventually degraded upon IL-1 stimulation. Although a point mutation changing lysine 134 to arginine (K134R) in IRAK abolished IL-1-induced IRAK ubiquitination and degradation, mutations of serines and threonines adjacent to lysine 134 to alanines ((S/T)A (131-144)) reduced IL-1-induced IRAK phosphorylation and abolished IRAK ubiquitination. Through the study of these IRAK modification mutants, we uncovered two parallel IL-1-mediated signaling pathways for NFkappaB activation, TAK1-dependent and MEKK3-dependent, respectively. These two pathways bifurcate at the level of IRAK modification. The TAK1-dependent pathway leads to IKKalpha/beta phosphorylation and IKKbeta activation, resulting in classical NFkappaB activation through IkappaBalpha phosphorylation and degradation. The TAK1-independent MEKK3-dependent pathway involves IKKgamma phosphorylation and IKKalpha activation, resulting in NFkappaB activation through IkappaBalpha phosphorylation and subsequent dissociation from NFkappaB but without IkappaBalpha degradation. These results provide significant insight to our further understanding of NFkappaB activation pathways.