J Immunol. 2013 Oct 1;191(7):3867-75

Barrio L, Saez de Guinoa J, Carrasco YR.

B cells use a plethora of TLR to recognize pathogen-derived ligands. These innate signals have an important function in the B cell adaptive immune response and modify their trafficking and tissue location. The direct role of TLR signaling on B cell dynamics nonetheless remains almost entirely unknown.

In this study, we used a state-of-the-art two-dimensional model combined with real-time microscopy to study the effect of TLR4 stimulation on mouse B cell motility in response to chemokines. We show that a minimum stimulation period is necessary for TLR4 modification of B cell behavior. TLR4 stimulation increased B cell polarization, migration, and directionality; these increases were dependent on the MyD88 signaling pathway and did not require ERK or p38 MAPK activity downstream of TLR4. In addition, TLR4 stimulation enhanced Rac GTPase activity and promoted sustained Rac activation in response to chemokines. These results increase our understanding of the regulation of B cell dynamics by innate signals and the underlying molecular mechanisms.

PLoS One. 2013 Aug 22;8(8):e72960

Zotes TM, Spada R, Mulens V, Pérez-Yagüe S, Sorzano CO, Okkenhaug K, Carrera AC, Barber DF.

The role of p110δ PI3K in lymphoid cells has been studied extensively, showing its importance in immune cell differentiation, activation and development. Altered T cell localization in p110δ-deficient mouse spleen suggested a role for p110δ in non-hematopoietic stromal cells, which maintain hematopoietic cell segregation.

We tested this hypothesis using p110δ^WT/WT mouse bone marrow to reconstitute lethally irradiated p110δ^WT/WT or p110δ^D910A/D910A (which express catalytically inactive p110δ) recipients, and studied localization, number and percentage of hematopoietic cell subsets in spleen and lymph nodes, in homeostatic conditions and after antigen stimulation. These analyses showed diffuse T cell areas in p110δ^D910A/D910A and in reconstituted p110δ^D910A/D910A mice in homeostatic conditions. In these mice, spleen CD4⁺ and CD8⁺ T cell numbers did not increase in response to antigen, suggesting that a p110δ^D910A/D910A stroma defect impedes correct T cell response. FACS analysis of spleen stromal cell populations showed a decrease in the percentage of gp38⁻CD31⁺ cells in p110δ^D910A/D910A mice. qRT-PCR studies detected p110δ mRNA expression in p110δ^WT/WT spleen gp38⁻CD31⁺ and gp38⁺CD31⁺ subsets, which was reduced in p110δ^D910A/D910A spleen. Lack of p110δ activity in these cell populations correlated with lower LTβR, CCL19 and CCL21 mRNA levels; these molecules participate in T cell localization to specific spleen areas. Our results could explain the lower T cell numbers and more diffuse T cell areas found in p110δ^D910A/D910A mouse spleen, as well as the lower T cell expansion after antigen stimulation in p110δ^D910A/D910A compared with p110δ^WT/WT mice.

PLoS One. 2013 Aug 22;8(8):e72674

Zotes TM, Arias CF, Fuster JJ, Spada R, Pérez-Yagüe S, Hirsch E, Wymann M, Carrera AC, Andrés V, Barber DF.

Atherosclerosis is an inflammatory disease regulated by infiltrating monocytes and T cells, among other cell types. Macrophage recruitment to atherosclerotic lesions is controlled by monocyte infiltration into plaques. Once in the lesion, macrophage proliferation in situ, apoptosis, and differentiation to an inflammatory (M1) or anti-inflammatory phenotype (M2) are involved in progression to advanced atherosclerotic lesions.

We studied the role of phosphoinositol-3-kinase (PI3K) p110γ in the regulation of in situ apoptosis, macrophage proliferation and polarization towards M1 or M2 phenotypes in atherosclerotic lesions. We analyzed atherosclerosis development in LDLR^−/−p110γ^+/− and LDLR^−/−p110γ^−/− mice, and performed expression and functional assays in tissues and primary cells from these and from p110γ^+/− and p110γ^−/− mice. Lack of p110γ in LDLR^−/− mice reduces the atherosclerosis burden. Atherosclerotic lesions in fat-fed LDLR^−/−p110γ^−/− mice were smaller than in LDLR^−/−p110γ^+/− controls, which coincided with decreased macrophage proliferation in LDLR^−/−p110γ^−/− mouse lesions. This proliferation defect was also observed in p110γ^−/− bone marrow-derived macrophages (BMM) stimulated with macrophage colony-stimulating factor (M-CSF), and was associated with higher intracellular cyclic adenosine monophosphate (cAMP) levels. In contrast, T cell proliferation was unaffected in LDLR^−/−p110γ^−/− mice. Moreover, p110γ deficiency did not affect macrophage polarization towards the M1 or M2 phenotypes or apoptosis in atherosclerotic plaques, or polarization in cultured BMM.

Our results suggest that higher cAMP levels and the ensuing inhibition of macrophage proliferation contribute to atheroprotection in LDLR^−/− mice lacking p110γ. Nonetheless, p110γ deletion does not appear to be involved in apoptosis, in macrophage polarization or in T cell proliferation.

J Clin Endocrinol Metab. 2013 Jul;98(7):2811-21

Rodríguez-Rodero S, Fernández AF, Fernández-Morera JL, Castro-Santos P, Bayon GF, Ferrero C, Urdinguio RG, Gonzalez-Marquez R, Suarez C, Fernández-Vega I, Fresno Forcelledo MF, Martínez-Camblor P, Mancikova V, Castelblanco E, Perez M, Marrón PI, Mendiola M, Hardisson D, Santisteban P, Riesco-Eizaguirre G, Matías-Guiu X, Carnero A, Robledo M, Delgado-Álvarez E, Menéndez-Torre E, Fraga MF.

Objective: The purpose of this study was to determine the global patterns of aberrant DNA methylation in thyroid cancer.

Research Design and Methods: We have used DNA methylation arrays to determine, for the first time, the genome-wide promoter methylation status of papillary, follicular, medullary, and anaplastic thyroid tumors.

Results: We identified 262 and 352 hypermethylated and 13 and 21 hypomethylated genes in differentiated papillary and follicular tumors, respectively. Interestingly, the other tumor types analyzed displayed more hypomethylated genes (280 in anaplastic and 393 in medullary tumors) than aberrantly hypermethylated genes (86 in anaplastic and 131 in medullary tumors). Among the genes indentified, we show that 4 potential tumor suppressor genes (ADAMTS8, HOXB4, ZIC1, and KISS1R) and 4 potential oncogenes (INSL4, DPPA2, TCL1B, and NOTCH4) are frequently regulated by aberrant methylation in primary thyroid tumors. In addition, we show that aberrant promoter hypomethylation-associated overexpression of MAP17 might promote tumor growth in thyroid cancer.

Conclusions: Thyroid cancer subtypes present differential promoter methylation signatures, and nondifferentiated subtypes are characterized by aberrant promoter hypomethylation rather than hypermethylation. Additional studies are needed to determine the potential clinical interest of the tumor subtype-specific DNA methylation signatures described herein and the role of aberrant promoter hypomethylation in nondifferentiated thyroid tumors.

J Mol Med. 2013 Aug;91(8):939-50

Huidobro C, Toraño EG, Fernández AF, Urdinguio RG, Rodríguez RM, Ferrero C, Martínez-Camblor P, Boix L, Bruix J, García-Rodríguez JL, Varela-Rey M, Mato JM, Martínez-Chantar ML, Fraga MF.

The basic mechanisms underlying promoter DNA hypermethylation in cancer are still largely unknown. It has been proposed that the levels of the methyl donor group in DNA methylation reactions, S-adenosylmethionine (SAMe), might be involved. SAMe levels depend on the glycine-N-methyltransferase (GNMT), a one-carbon group methyltransferase, which catalyzes the conversion of SAMe to S-adenosylhomocysteine in hepatic cells. GNMT has been proposed to display tumor suppressor activity and to be frequently repressed in hepatocellular carcinoma (HCC).

In this study, we show that GNMT shows aberrant DNA hypermethylation in some HCC cell lines and primary tumors (20 %). GNMT hypermethylation could contribute to gene repression and its restoration in cell lines displaying hypermethylation-reduced tumor growth in vitro. In agreement, human primary tumors expressing GNMT were of smaller size than tumors showing GNMT hypermethylation. Genome-wide analyses of gene promoter methylation identified 277 genes whose aberrant methylation in HCC was associated with GNMT methylation/expression. The findings in this manuscript indicate that DNA hypermethylation plays an important role in the repression of GNMT in HCC and that loss of GNMT in human HCC could promote the establishment of aberrant DNA methylation patterns at specific gene promoters.

Cancer Cell. 2013 Jul 8;24(1):15-29

Rad R, Cadiñanos J, Rad L, Varela I, Strong A, Kriegl L, Constantino-Casas F, Eser S, Hieber M, Seidler B, Price S, Fraga MF, Calvanese V, Hoffman G, Ponstingl H, Schneider G, Yusa K, Grove C, Schmid RM, Wang W, Vassiliou G, Kirchner T, McDermott U, Liu P, Saur D, Bradley A.

We show that BRAF^V600E initiates an alternative pathway to colorectal cancer (CRC), which progresses through a hyperplasia/adenoma/carcinoma sequence. This pathway underlies significant subsets of CRCs with distinctive pathomorphologic/genetic/epidemiologic/clinical characteristics.

Genetic and functional analyses in mice revealed a series of stage-specific molecular alterations driving different phases of tumor evolution and uncovered mechanisms underlying this stage specificity. We further demonstrate dose-dependent effects of oncogenic signaling, with physiologic Braf^V600E expression being sufficient for hyperplasia induction, but later stage intensified Mapk-signaling driving both tumor progression and activation of intrinsic tumor suppression. Such phenomena explain, for example, the inability of p53 to restrain tumor initiation as well as its importance in invasiveness control, and the late stage specificity of its somatic mutation. Finally, systematic drug screening revealed sensitivity of this CRC subtype to targeted therapeutics, including Mek or combinatorial PI3K/Braf inhibition.

Environ Health Perspect. 2013 Jun;121(6):650-6

Tajuddin SM, Amaral AF, Fernández AF, Rodríguez-Rodero S, Rodríguez RM, Moore LE, Tardón A, Carrato A, García-Closas M, Silverman DT, Jackson BP, García-Closas R, Cook AL, Cantor KP, Chanock S, Kogevinas M, Rothman N, Real FX, Fraga MF, Malats N; Spanish Bladder Cancer/EPICURO Study Investigators.

BACKGROUND: Altered DNA methylation has been associated with various diseases.

OBJECTIVE: We evaluated the association between levels of methylation in leukocyte DNA at long interspersed nuclear element 1 (LINE-1) and genetic and non-genetic characteristics of 892 control participants from the Spanish Bladder Cancer/EPICURO study.

METHODS: We determined LINE-1 methylation levels by pyrosequencing. Individual data included demographics, smoking status, nutrient intake, toenail concentrations of 12 trace elements, xenobiotic metabolism gene variants, and 515 polymorphisms among 24 genes in the one-carbon metabolism pathway. To assess the association between LINE-1 methylation levels (percentage of methylated cytosines) and potential determinants, we estimated beta coefficients (βs) by robust linear regression.

RESULTS: Women had lower levels of LINE-1 methylation than men (β = -0.7, p = 0.02). Persons who smoked blond tobacco showed lower methylation than nonsmokers (β = -0.7, p = 0.03). Arsenic toenail concentration was inversely associated with LINE-1 methylation (β = -3.6, p = 0.003). By contrast, iron (β = 0.002, p = 0.009) and nickel (β = 0.02, p = 0.004) were positively associated with LINE-1 methylation. Single nucleotide polymorphisms (SNPs) in DNMT3A (rs7581217-per allele, β = 0.3, p = 0.002), TCN2 (rs9606756-GG, β = 1.9, p = 0.008; rs4820887-AA, β = 4.0, p = 4.8 × 10-7; rs9621049-TT, β = 4.2, p = 4.7 × 10-9), AS3MT (rs7085104-GG, β = 0.7, p = 0.001), SLC19A1 (rs914238, TC vs. TT: β = 0.5 and CC vs. TT: β = -0.3, global p = 0.0007) and MTHFS (rs1380642, CT vs. CC: β = 0.3 and TT vs. CC; β = -0.8, global p = 0.05) were associated with LINE-1 methylation.

CONCLUSIONS: We identified several characteristics, environmental factors, and common genetic variants that predicted DNA methylation among study participants.

Environ Microbiol. 2013 Aug 1

Natale P, Pazos M, Vicente M.

Septation in Escherichia coli involves complex molecular mechanisms that contribute to the accuracy of bacterial division. The proto-ring, a complex made up by the FtsZ, FtsA and ZipA proteins, forms at the beginning of the process and directs the assembly of the full divisome. Central to this complex is the FtsZ protein, a GTPase able to assemble into a ring-like structure that responds to several modulatory inputs including mechanisms to position the septum at midcell.

The connection with the cell wall synthesising machinery stabilizes the constriction of the cytoplasmic membrane. Although a substantial amount of evidence supports this description, many details on how individual divisome elements are structured or how they function are subjected to controversial interpretations. We discuss these discrepancies arising from incomplete data and from technical difficulties imposed by the small size of bacteria. Future work, including more powerful imaging and reconstruction technologies, will help to clarify the missing details on the architecture and function of the bacterial division machinery.

Virology. 2013 Aug 1;442(2):122-31

Pérez Jde J, Udeshi ND, Shabanowitz J, Ciordia S, Juárez S, Scott CL, Olszewski NE, Hunt DF, García JA.

O-GlcNAcylation is a dynamic protein modification which has been studied mainly in metazoans. We reported previously that an Arabidopsis thaliana O-GlcNAc transferase modifies at least two threonine residues of the Plum pox virus (PPV) capsid protein (CP).

Now, six additional residues were shown to be involved in O-GlcNAc modification of PPV CP. CP O-GlcNAcylation was abolished in the PPV CP7-T/A mutant, in which seven threonines were mutated. PPV CP7-T/A infected Nicotiana clevelandii, Nicotiana benthamiana, and Prunus persica without noticeable defects. However, defects in infection of A. thaliana were readily apparent. In mixed infections of wild-type arabidopsis, the CP7-T/A mutant was outcompeted by wild-type virus. These results indicate that CP O-GlcNAcylation has a major role in the infection process. O-GlcNAc modification may have a role in virion assembly and/or stability as the CP of PPV CP7-T/A was more sensitive to protease digestion than that of the wild-type virus.

Environ Microbiol. 2013 Jul 25. doi: 10.1111/1462-2920.12224

Nikel PI, Kim J, de Lorenzo V.

While the natural niches of the soil bacterium Pseudomonas putida are unlikely to include significant amounts of free glycerol as a growth substrate, this bacterium is genetically equipped with the functions required for its metabolism. We have resorted to deep sequencing of the transcripts in glycerol-grown P. putida KT2440 cells to gain an insight into the biochemical and regulatory components involved in the shift between customary C sources (e.g. glucose or succinate) to the polyol.

Transcriptomic results were contrasted with key enzymatic activities under the same culture conditions. Cognate expression profiles revealed that genes encoding enzymes of the Entner–Doudoroff route and other catabolic pathways, e.g. the gluconate and 2-ketogluconate loops, were significantly downregulated on glycerol. Yet, the compound simultaneously elicited a gluconeogenic response that indicated an efficient channelling of C skeletons back to biomass build-up through the glyoxylate shunt rather than energization of the cells through downwards pathways, i.e. tricarboxylic acid cycle and oxidative phosphorylation. The simultaneous glycolytic and gluconeogenic metabolic regimes on glycerol, paradoxical as they seem, make sense from an ecological point of view by favouring prevalence versus exploration. This metabolic situation was accompanied by a considerably low expression of stress markers as compared with other C sources.