Early growth response 2 (Egr-2) expression is triggered by NF-κB activation.

Transcription factors are known to play multiple roles in cellular function. Investigators report that factors such as early growth response (Egr) protein and nuclear factor kappa B (NF-κB) are activated in the brain during cancer, brain injury, inflammation, and/or memory. To explore NF-κB activity further, we investigated the transcriptomes of hippocampal slices following electrical stimulation of NF-κB p50 subunit knockout mice (p50-/-) versus their controls (p50+/+). We found that the early growth response gene Egr-2 was upregulated by NF-κB activation, but only in p50+/+ hippocampal slices. We then stimulated HeLa cells and primary cortical neurons with tumor necrosis factor alpha (TNFα) to activate NF-κB and increase the expression of Egr-2. The Egr-2 promoter sequence was analyzed for NF-κB binding sites and chromatin immunoprecipitation (ChIP) assays were performed to confirm promoter occupancy in vivo. We discovered that NF-κB specifically binds to an NF-κB consensus binding site within the proximal promoter region of Egr-2. Luciferase assay demonstrated that p50 was able to transactivate the Egr-2 promoter in vitro. Small interfering RNA (siRNA)-mediated p50 knockdown corroborated other Egr-2 expression studies. We show for the first time a novel link between NF-κB activation and Egr-2 expression with Egr-2 expression directly controlled by the transcriptional activity of NF-κB.

Interactions between Arabidopsis DNA repair genes UVH6, DDB1A, and DDB2 during abiotic stress tolerance and floral development.

Plants must protect themselves from a spectrum of abiotic stresses. For example, the sun is a source of heat, intense light, and DNA-damaging ultraviolet (UV) rays. Damaged DNA binding protein 1A (DDB1A), DDB2, and UV hypersensitive 6 (UVH6)/XPD are all involved in the repair of UV-damaged DNA - DDB1A and DDB2 in the initial damage recognition stage, while the UVH6/XPD helicase unwinds the damaged strand. We find that, as predicted, Arabidopsis ddb1a and ddb2 mutants do not affect uvh6/xpd UV tolerance. In addition, uvh6 is heat sensitive, and ddb1a and ddb2 weakly enhance this trait. The uvh6 ddb1a and uvh6 ddb2 double mutants also exhibit sensitivity to oxidative stress, suggesting a role for DDB1 complexes in heat and oxidative stress tolerance. Finally, we describe a new uvh6 phenotype, the low penetrance production of flowers with five petals and five sepals. ddb1a and ddb2 suppress this phenotype in uvh6 mutants. Interestingly, heat treatment also induces five-petalled flowers in the ddb1a and ddb2 single mutants. Thus UVH6, DDB1A, and DDB2 all contribute to UV tolerance, heat tolerance and floral patterning.

Genetic interactions between Arabidopsis DET1 and UVH6 during development and abiotic stress response.

Plants must adapt to a variety of abiotic inputs, including visible light, ultraviolet (UV) light, and heat. In Arabidopsis thaliana, DE-ETIOLATED 1 (DET1) plays a role in visible light signaling, UV tolerance, and development. UV-HYPERSENSITIVE 6 (UVH6) mutants are UV and heat sensitive, as well as dwarf and pale, like det1. In this study, we examine the genetic interactions between these two genes. In dark-grown seedlings, uvh6 exhibits a weak de-etiolated phenotype but does not affect the stronger de-etiolated phenotype of det1. In the light, det1 is epistatic to uvh6 with regard to chlorophyll level, but their effect on all size parameters is additive and therefore independent. With regard to UV tolerance, det1 UV resistance is epistatic to uvh6 UV sensitivity. In heat stress experiments, det1 enhances heat-induced tissue damage in the uvh6 background but suppresses heat-induced growth inhibition. Thus, det1 acts epistatically to uvh6 with respect to de-etiolation, chlorophyll level, UV tolerance, and heat-induced growth inhibition, whereas det1 and uvh6 act additively to regulate plant size and heat-induced cell death. These data provide insight into interplay between light and heat signaling.

NF-κB p50 subunit knockout impairs late LTP and alters long term memory in the mouse hippocampus.

BACKGROUND: Nuclear factor kappa B (NF-κB) is a transcription factor typically expressed with two specific subunits (p50, p65). Investigators have reported that NF-κB is activated during the induction of in vitro long term potentiation (LTP), a paradigm of synaptic plasticity and correlate of memory, suggesting that NF-κB may be necessary for some aspects of memory encoding. Furthermore, NF-κB has been implicated as a potential requirement in behavioral tests of memory. Unfortunately, very little work has been done to explore the effects of deleting specific NF-κB subunits on memory. Studies have shown that NF-κB p50 subunit deletion (p50-/-) leads to memory deficits, however some recent studies suggest the contrary where p50-/- mice show enhanced memory in the Morris water maze (MWM). To more critically explore the role of the NF-κB p50 subunit in synaptic plasticity and memory, we assessed long term spatial memory in vivo using the MWM, and synaptic plasticity in vitro utilizing high frequency stimuli capable of eliciting LTP in slices from the hippocampus of NF-κB p50-/- versus their controls (p50+/+). RESULTS: We found that the lack of the NF-κB p50 subunit led to significant decreases in late LTP and in selective but significant alterations in MWM tests (i.e., some improvements during acquisition, but deficits during retention). CONCLUSIONS: These results support the hypothesis that the NF-κ p50 subunit is required in long term spatial memory in the hippocampus.

Tyrosine kinase-independent activation of extracellular-regulated kinase (ERK) 1/2 by the insulin-like growth factor-1 receptor.

The extracellular-regulated kinase (ERK1/2) is a key conduit for transduction of signals from growth factor receptors to the nucleus. Previous work has shown that ERK1/2 activation in response to IGF-1 may require the participation of G proteins, but the role of the receptor tyrosine kinase in this process has not been clearly resolved. This investigation of IGF-1 receptor function was therefore designed to examine the contribution of the receptor tyrosine kinase to ERK1/2 activation. Phosphorylation of ERK1/2 in smooth muscle cells following treatment with IGF-1 was not blocked by pretreatment with AG1024 or picropodophylin, inhibitors of the IGF-1 receptor tyrosine kinase. Likewise, IGF-1 activated ERK1/2 in cells expressing a kinase-dead mutant of the IGF-1 receptor. ERK1/2 activation was unaffected by the phosphatidylinositol 3-kinase inhibitor LY-294002, but was sensitive to inhibitors of Src kinase, phospholipase C and Gßγ subunit signalling. Treatment with αIR-3, a neutralizing monoclonal antibody, also stimulated ERK1/2 phosphorylation without concomitant activation of the receptor tyrosine kinase. Phosphoprotein mapping of IGF-1 and αIR-3 treated cells confirmed that antibody-induced ERK1/2 phosphorylation occurred in the absence of tyrosine kinase phosphorylation, and enabled extension of these findings to p38 MAPK. These results suggest that stimulation of ERK1/2 phosphorylation by IGF-1 does not require activation of the receptor tyrosine kinase.

Vitamin C restores healthy aging in a mouse model for Werner syndrome.

Werner syndrome (WS) is a premature aging disorder caused by mutations in a RecQ-like DNA helicase. Mice lacking the helicase domain of the WRN homologue exhibit many phenotypic features of WS, including a prooxidant status and a shorter mean life span compared to wild-type animals. Here, we show that Wrn mutant mice also develop premature liver sinusoidal endothelial defenestration along with inflammation and metabolic syndrome. Vitamin C supplementation rescued the shorter mean life span of Wrn mutant mice and reversed several age-related abnormalities in adipose tissues and liver endothelial defenestration, genomic integrity, and inflammatory status. At the molecular level, phosphorylation of age-related stress markers like Akt kinase-specific substrates and the transcription factor NF-kappaB, as well as protein kinase Cdelta and Hif-1alpha transcription factor levels, which are increased in the liver of Wrn mutants, were normalized by vitamin C. Vitamin C also increased the transcriptional regulator of lipid metabolism PPARalpha. Finally, microarray and gene set enrichment analyses on liver tissues revealed that vitamin C decreased genes normally up-regulated in human WS fibroblasts and cancers, and it increased genes involved in tissue injury response and adipocyte dedifferentiation in obese mice. Vitamin C did not have such effect on wild-type mice. These results indicate that vitamin C supplementation could be beneficial for patients with WS.