The Transcription Factor NFAT5 Is Required for Cyclin Expression and Cell Cycle Progression in Cells Exposed to Hypertonic Stress

Background: Hypertonicity can perturb cellular functions, induce DNA damage-like responses and inhibit proliferation. The transcription factor NFAT5 induces osmoprotective gene products that allow cells to adapt to sustained hypertonic conditions. Although it is known that NFAT5-deficient lymphocytes and renal medullary cells have reduced proliferative capacity and viability under hypertonic stress, less is understood about the contribution of this factor to DNA damage responses and cell cycle regulation. Methodology/Principal Findings: We have generated conditional knockout mice to obtain NFAT5−/− T lymphocytes, which we used as a model of proliferating cells to study NFAT5-dependent responses. We show that hypertonicity triggered an early, NFAT5-independent, genotoxic stress-like response with induction of p53, p21 and GADD45, downregulation of cyclins, and cell cycle arrest. This was followed by an NFAT5-dependent adaptive phase in wild-type cells, which induced an osmoprotective gene expression program, downregulated stress markers, resumed cyclin expression and proliferation, and displayed enhanced NFAT5 transcriptional activity in S and G2/M. In contrast, NFAT5−/− cells failed to induce osmoprotective genes and exhibited poorer viability. Although surviving NFAT5−/− cells downregulated genotoxic stress markers, they underwent cell cycle arrest in G1/S and G2/M, which was associated with reduced expression of cyclins E1, A2 and B1. We also show that pathologic hypertonicity levels, as occurring in plasma of patients and animal models of osmoregulatory disorders, inhibited the induction of cyclins and aurora B kinase in response to T cell receptor stimulation in fresh NFAT5−/− lymphocytes. Conclusions/Significance: We conclude that NFAT5 facilitates cell proliferation under hypertonic conditions by inducing an osmoadaptive response that enables cells to express fundamental regulators needed for cell cycle progression.Molecular and Cellular Biolog

Conjugative Plasmids of Neisseria gonorrhoeae

Many clinical isolates of the human pathogen Neisseria gonorrhoeae contain conjugative plasmids. The host range of these plasmids is limited to Neisseria species, but presence of a tetracycline (tetM) determinant inserted in several of these plasmids is an important cause of the rapid spread of tetracycline resistance. Previously plasmids with different backbones (Dutch and American type backbones) and with and without different tetM determinants (Dutch and American type tetM determinants) have been identified. Within the isolates tested, all plasmids with American or Dutch type tetM determinants contained a Dutch type plasmid backbone. This demonstrated that tetM determinants should not be used to differentiate between conjugal plasmid backbones. The nucleotide sequences of conjugative plasmids with Dutch type plasmid backbones either not containing the tetM determinant (pEP5233) or containing Dutch (pEP5289) or American (pEP5050) type tetM determinants were determined. Analysis of the backbone sequences showed that they belong to a novel IncP1 subfamily divergent from the IncP1α, β, γ, δ and ε subfamilies. The tetM determinants were inserted in a genetic load region found in all these plasmids. Insertion was accompanied by the insertion of a gene with an unknown function, and rearrangement of a toxin/antitoxin gene cluster. The genetic load region contains two toxin/antitoxins of the Zeta/Epsilon toxin/antitoxin family previously only found in Gram positive organisms and the virulence associated protein D of the VapD/VapX toxin/antitoxin family. Remarkably, presence of VapX of pJD1, a small cryptic neisserial plasmid, in the acceptor strain strongly increased the conjugation efficiency, suggesting that it functions as an antitoxin for the conjugative plasmid. The presence of the toxin and antitoxin on different plasmids might explain why the host range of this IncP1 plasmid is limited to Neisseria species. The isolated plasmids conjugated efficiently between N. gonorrhoeae strains, but did not enhance transfer of a genetic marker

The p53 Inhibitor MDM2 Facilitates Sonic Hedgehog-Mediated Tumorigenesis and Influences Cerebellar Foliation

Disruption of cerebellar granular neuronal precursor (GNP) maturation can result in defects in motor coordination and learning, or in medulloblastoma, the most common childhood brain tumor. The Sonic Hedgehog (Shh) pathway is important for GNP proliferation; however, the factors regulating the extent and timing of GNP proliferation, as well as GNP differentiation and migration are poorly understood. The p53 tumor suppressor has been shown to negatively regulate the activity of the Shh effector, Gli1, in neural stem cells; however, the contribution of p53 to the regulation of Shh signaling in GNPs during cerebellar development has not been determined. Here, we exploited a hypomorphic allele of Mdm2 (Mdm2puro), which encodes a critical negative regulator of p53, to alter the level of wild-type MDM2 and p53 in vivo. We report that mice with reduced levels of MDM2 and increased levels of p53 have small cerebella with shortened folia, reminiscent of deficient Shh signaling. Indeed, Shh signaling in Mdm2-deficient GNPs is attenuated, concomitant with decreased expression of the Shh transducers, Gli1 and Gli2. We also find that Shh stimulation of GNPs promotes MDM2 accumulation and enhances phosphorylation at serine 166, a modification known to increase MDM2-p53 binding. Significantly, loss of MDM2 in Ptch1+/− mice, a model for Shh-mediated human medulloblastoma, impedes cerebellar tumorigenesis. Together, these results place MDM2 at a major nexus between the p53 and Shh signaling pathways in GNPs, with key roles in cerebellar development, GNP survival, cerebellar foliation, and MB tumorigenesis

Precancerous Stem Cells Have the Potential for both Benign and Malignant Differentiation

Cancer stem cells (CSCs) have been identified in hematopoietic and solid tumors. However, their precursors—namely, precancerous stem cells (pCSCs) —have not been characterized. Here we experimentally define the pCSCs that have the potential for both benign and malignant differentiation, depending on environmental cues. While clonal pCSCs can develop into various types of tissue cells in immunocompetent mice without developing into cancer, they often develop, however, into leukemic or solid cancers composed of various types of cancer cells in immunodeficient mice. The progress of the pCSCs to cancers is associated with the up-regulation of c-kit and Sca-1, as well as with lineage markers. Mechanistically, the pCSCs are regulated by the PIWI/AGO family gene called piwil2. Our results provide clear evidence that a single clone of pCSCs has the potential for both benign and malignant differentiation, depending on the environmental cues. We anticipate pCSCs to be a novel target for the early detection, prevention, and therapy of cancers

The Genome of a Pathogenic Rhodococcus: Cooptive Virulence Underpinned by Key Gene Acquisitions

We report the genome of the facultative intracellular parasite Rhodococcus equi, the only animal pathogen within the biotechnologically important actinobacterial genus Rhodococcus. The 5.0-Mb R. equi 103S genome is significantly smaller than those of environmental rhodococci. This is due to genome expansion in nonpathogenic species, via a linear gain of paralogous genes and an accelerated genetic flux, rather than reductive evolution in R. equi. The 103S genome lacks the extensive catabolic and secondary metabolic complement of environmental rhodococci, and it displays unique adaptations for host colonization and competition in the short-chain fatty acid–rich intestine and manure of herbivores—two main R. equi reservoirs. Except for a few horizontally acquired (HGT) pathogenicity loci, including a cytoadhesive pilus determinant (rpl) and the virulence plasmid vap pathogenicity island (PAI) required for intramacrophage survival, most of the potential virulence-associated genes identified in R. equi are conserved in environmental rhodococci or have homologs in nonpathogenic Actinobacteria. This suggests a mechanism of virulence evolution based on the cooption of existing core actinobacterial traits, triggered by key host niche–adaptive HGT events. We tested this hypothesis by investigating R. equi virulence plasmid-chromosome crosstalk, by global transcription profiling and expression network analysis. Two chromosomal genes conserved in environmental rhodococci, encoding putative chorismate mutase and anthranilate synthase enzymes involved in aromatic amino acid biosynthesis, were strongly coregulated with vap PAI virulence genes and required for optimal proliferation in macrophages. The regulatory integration of chromosomal metabolic genes under the control of the HGT–acquired plasmid PAI is thus an important element in the cooptive virulence of R. equi