1,25-Dihydroxyvitamin D [1,25(OH)2D3] is known to have anti-inflammatory activity; however, the molecular mechanism remains poorly defined. Here we show that the nuclear vitamin D receptor (VDR) is directly involved in the regulation of NF-κB activation, a pathway essential for inflammatory response. In mouse embryonic fibroblasts (MEFs) derived from VDR−/− mice, the basal level of κB inhibitor (IκB) α protein was markedly decreased compared with VDR+/− MEFs; however, degradation of IκBα and its phosphorylation in response to TNF-α treatment or Salmonella infection were not altered in VDR−/− cells, neither were the levels of IκB kinase-α and IκB kinase-β proteins. Consistent with IκBα reduction, p65 accumulation in the nucleus was markedly increased in unstimulated VDR−/− cells. In addition, the physical interaction between VDR and p65 was absent in VDR−/− MEFs, which may free p65 and increase its activity. Consequently, these alterations combined led to a marked increase in nuclear p65 DNA binding and NF-κB transcriptional activity; consistently, induction of IL-6 by TNF-α or IL-1β was much more robust in VDR−/− than in VDR+/− cells, indicating that VDR−/− cells are more susceptible to inflammatory stimulation. Therefore, cells lacking VDR appear to be more proinflammatory due to the intrinsic high NF-κB activity. The reduction of IκBα in VDR−/− MEFs may be partially explained by the lack of VDR-mediated stabilization of IκBα by 1,25(OH)2D3. This is supported by the observation that IκBα degradation induced by TNF-α was inhibited by 1,25(OH)2D3 in VDR+/− cells, but not in VDR−/− cells. Taken together, these data suggest that VDR plays an inhibitory role in the regulation of NF-κB activation.

Wild-type (WT) Salmonella typhimurium causes acute intestinal inflammation by activating the nuclear factor kappa B (NF-kB) pathway. Interestingly, WT Salmonella infection also causes degradation of b-catenin, a regulator of cellular proliferation. Regulation of b-catenin and the inhibitor of NF-kB, IkBa, is strikingly similar, involving phosphorylation at identical sites, ubiquitination by the same E3 ligase, and subsequent proteasomal degradation. However, how b-catenin directly regulates the NF-kB pathway during bacteria-induced inflammation in vivo is unknown. Using streptomycinpretreated mice challenged with Salmonella, we demonstrated that WT Salmonella stimulated b-catenin degradation and decreased the physical association between NF-kB and b-catenin. Accordingly, WT Salmonella infection decreased the expression of c-myc, a b-catenin-regulated target gene, and increased the levels of IL-6 and TNF-a, the NF-kB-regulated target genes. Bacterial infection directly stimulated phosphorylation of b-catenin, both in vivo and in vitro. Closer examination revealed that glycogen synthase kinase 3b (GSK-3b) kinase activity was increased in response to WT Salmonella, whereas non-virulent Salmonella had no effect. siRNA of GSK-3b was able to stabilize IkBa in response to WT Salmonella. Pretreatment for 24 h with LiCl, an inhibitor of GSK-3b, reduced WT Salmonella induced IL-8 secretion. Additionally, cells expressing constitutively active b-catenin showed IkBa stabilization and inhibition of NF-kB activity not only after WT Salmonella infection but also after commensal bacteria (Escherichia coli F18) and TNF-a treatment. This study suggests a new role for b-catenin as a negative regulator of inflammation. Bacteria pathogenicity requires overcoming or altering many very effective host defense mechanisms, 1 including the activation of nuclear factor kappa B (NF-kB). 2-4 Bacterial invasion of intestinal epithelial cells (IECs) stimulates NF-kB and, interestingly, degradation of b-catenin, 5 a potent transcriptional factor responsible for cellular proliferation and differentiation. It is notable that regulation of b-catenin and the inhibitor of NF-kB, IkBa, are strikingly similar, involving phosphorylation of the same N-terminal serine sequence sites, ubiquitination by the same E3 ligase complex, and subsequent proteasomal degradation. 6,7 To date, no publications have reported on the physiological significance of b-catenin's potential inter-relationship with the NF-kB inflammatory pathway after bacterial infection in vivo.In this study, therefore, we investigated the role of b-catenin in modulating the proinflammatory response mediated by NF-kB subsequent to Salmonella infection in vivo. We examined the possibility that b-catenin functions as a negative regulator of NF-kB, much in the same way as IkBa, through physical interaction with NF-kB. Additionally, we established the role of glycogen synthase kinase 3b (GSK-3b), as the negative regulator of b-catenin's stability and subsequent reactivity with NF-kB in both in vit...

In the adult mammalian CNS, the growth inhibitors oligodendrocyte-myelin glycoprotein (OMgp) and the reticulon RTN4 (Nogo) are broadly expressed in oligodendrocytes and neurons. Nogo and OMgp complex with the neuronal cell surface receptors Nogo receptor-1 (NgR1) and paired Ig-like receptor-B (PirB) to regulate neuronal morphology. In the healthy CNS, NgR1 regulates dendritic spine shape and attenuates activity-driven synaptic plasticity at Schaffer collateral-CA1 synapses. Here, we examine whether Nogo and OMgp influence functional synaptic plasticity, the efficacy by which synaptic transmission occurs. In acute hippocampal slices of adult mice, Nogo-66 and OMgp suppress NMDA receptor-dependent long-term potentiation (LTP) when locally applied to Schaffer collateral-CA1 synapses. Neither Nogo-66 nor OMgp influences basal synaptic transmission or paired-pulse facilitation, a form of short-term synaptic plasticity. PirB

Salmonella-epithelial cell interactions are known to activate the proinflammatory NF-kappaB signaling pathway and have recently been found to also influence the beta-catenin signaling pathway, an important regulator of epithelial cell proliferation and differentiation. Here, using polarized epithelial cell models, we demonstrate that these same bacteria-mediated effects also direct the molecular crosstalk between the NF-kappaB and beta-catenin signaling pathways. Convergence of these two pathways is a result of the direct interaction between the NF-kappaB p50 subunit and beta-catenin. We show that PhoP(c), the avirulent derivative of a wild-type Salmonella strain, attenuates NF-kappaB activity by stabilizing the association of beta-catenin with NF-kappaB. In cell lines expressing constitutively active beta-catenin, IkappaBalpha protein was indirectly stabilized and NF-kappaB activity was repressed after wild-type Salmonella colonization. Accordingly, constitutively active beta-catenin was found to inhibit the secretion of IL-8. Thus our findings strongly suggest that the crosstalk between the beta-catenin and NF-kappaB signaling pathways is an important regulator of intestinal inflammation.

Mitochondrial Ca2+ uptake and poly(ADP-ribose) polymerase-1 (PARP-1) activation are both required for glutamate-induced excitotoxic neuronal death. Since activation of the glutamate receptors can induce increased levels of reactive oxygen species (ROS), we investigated the relationship of mitochondrial Ca 2+ uptake and ROS generation, and the possibility that ROS increase is a required signal for PARP-1 activation in cultured striatal neurons. Based on the spatial profile of NMDA-induced ROS generation, we found that only mitochondria showed a significant ROS increase within 30 min after NMDA receptor activation. This ROS increase was inhibited by the mitochondrial complex inhibitors rotenone and oligomycin, but not by the cytosolic phospholipase A 2 or xanthine oxidase inhibitors. Mitochondrial ROS generation was also inhibited by both removal of Ca 2+ from extracellular medium and blockage of mitochondrial Ca 2+ uptake by either a mitochondrial uncoupler or a Ca 2+ uniporter inhibitor. Furthermore, both DNA damage and PARP-1 activation induced by NMDA treatment was inhibited by blocking mitochondrial Ca 2+ uptake or by antioxidants. Our results demonstrate that ROS production during the early stage of acute excitotoxicity derives primarily from mitochondria and is Ca 2+ -dependent. More importantly, the increase of mitochondrial ROS serves as a signal for PARP-1 activation, suggesting that concomitant mitochondrial Ca 2+ uptake and PARP-1 activation constitute a unified mechanism for excitotoxic neuronal death.

Titin is a very large alternatively spliced protein that performs multiple functions in heart and skeletal muscle. A rat strain is described with an autosomal dominant mutation that alters the isoform expression of titin. While wild type animals go through a developmental program where the 3.0 MDalton N2B becomes the major isoform expressed by two to three weeks after birth (~85%), the appearance of the N2B is markedly delayed in heterozygotes and never reaches more than 50% of the titin in the adult. Homozygote mutants express a giant titin of the N2BA isoform type (3.9 MDalton) that persists as the primary titin species through ages of more than one and half years. The mutation does not affect the isoform switching of troponin T, a protein that also is alternatively spliced with developmental changes. The basis for the apparently greater size of the giant titin in homozygous mutants was not determined, but additional length was not due to inclusion of sequence from larger numbers of PEVK exons or the Novex III exon. Passive tension measurements using isolated cardiomyocytes from homozygous mutants showed that cells could be stretched to sarcomere lengths greater than 4 µm without breakage. This novel rat model should be useful for exploring the potential role of titin in the Frank-Starling relationship and mechano-sensing/signaling mechanisms.

During animal development, blast cell lineages are generated by repeated divisions of a mother cell into a series of daughter cells, often with a specific series of distinct fates. Nanos is a translational regulator that is involved in germline development in diverse animals and also involved in somatic patterning in insects. Recently, Nanos was found to be required for maintenance of stem cell divisions in the Drosophila germline. We have found that in the mollusk Ilyanassa, Nanos messenger RNA and protein are specifically localized in the mesendodermal blast cell lineage derived from the strongly conserved 4d cell. Nanos activity is required for differentiation of multiple tissues that are derived from the 4d cell, showing that IoNanos is required for somatic development in this embryo. At the cellular level, we show that IoNanos activity is required for the highly stereotyped cleavage pattern of the 4d lineage, the proliferative capacity of the blast cells, and the marked asymmetry of the blast cell divisions. These results suggest that IoNanos is involved in regulating blast cell behaviors in the 4d lineage.

Human SEMAPHORIN 5A (SEMA5A) is an autism susceptibility gene; however, its function in brain development is unknown. In this study, we show that mouse Sema5A negatively regulates synaptogenesis in early, developmentally born, hippocampal dentate granule cells (GCs). Sema5A is strongly expressed by GCs and regulates dendritic spine density in a cell-autonomous manner. In the adult mouse brain, newly born Sema5A−/− GCs show an increase in dendritic spine density and increased AMPA-type synaptic responses. Sema5A signals through PlexinA2 co-expressed by GCs, and the PlexinA2-RasGAP activity is necessary to suppress spinogenesis. Like Sema5A−/− mutants, PlexinA2−/− mice show an increase in GC glutamatergic synapses, and we show that Sema5A and PlexinA2 genetically interact with respect to GC spine phenotypes. Sema5A−/− mice display deficits in social interaction, a hallmark of autism-spectrum-disorders. These experiments identify novel intra-dendritic Sema5A/PlexinA2 interactions that inhibit excitatory synapse formation in developmentally born and adult-born GCs, and they provide support for SEMA5A contributions to autism-spectrum-disorders.DOI: http://dx.doi.org/10.7554/eLife.04390.001