Die Rattenfänger von Balingen : wie die Bundeswehr mit Musik neuen Nachwuchs sucht

<div><p>Nuclear lamins are important structural and functional proteins in mammalian cells, but little is known about the mechanisms and cofactors that regulate their traffic into the nucleus. Here, we demonstrate that trafficking of lamin A, but not lamin B1, and its assembly into the nuclear envelope are regulated by sorting nexin 6 (SNX6), a major component of the retromer that targets proteins and other molecules to specific subcellular locations. SNX6 interacts with lamin A <i>in vitro</i> and <i>in vivo</i> and links it to the outer surface of the endoplasmic reticulum in human and mouse cells. SNX6 transports its lamin A cargo to the nuclear envelope in a process that takes several hours. Lamin A protein levels in the nucleus augment or decrease, respectively, upon gain or loss of SNX6 function. We further show that SNX6-dependent lamin A nuclear import occurs across the nuclear pore complex via a RAN-GTP-dependent mechanism. These results identify SNX6 as a key regulator of lamin A synthesis and incorporation into the nuclear envelope.</p></div

Extracellular PPi metabolism in MPiφs.

<p><b>(A)</b> mRNA levels for the indicated ectoenzymes in unpolarized M0φs and Pi-activated MPiφs. <b>(B)</b> Immunoblot analysis of the indicated ectoenzymes. Relative levels were quantified by densitometry and were normalized to α–tubulin. <b>(C)</b> eNTPD activity, quantified as PPi produced from hydrolysis of 1 μmol/L ATP in 1h. (<b>D</b>) Alkaline phosphatase activity. Statistical significance was determined by unpaired Student’s <i>t</i>-test. Results are presented as mean ± SE of 4 independent experiments with 3 mice per experiment. <b>ATP</b>, adenosine triphosphate; <b>AMP</b>, adenosine monophosphate; <b>PPi</b>, pyrophosphate; <b>Pi</b>, inorganic phosphate; <b>eNPP1</b>, ectoenzyme nucleotide pyrophosphatase/phosphodiesterase-1; <b>eNTPD1</b>, ectonucleoside triphosphate diphosphohydrolase 1; <b>TNAP</b>, tissue non-specific alkaline phosphatase. **, <i>P</i><0.01; ***, <i>P</i><0,001.</p

MPiφs prevent calcium-phosphate deposition by increasing extracellular ATP and PPi.

<p>Fixed VSMCs were incubated in pro-calcifying medium over 5 days with 0.4 μm polycarbonate transwells containing unpolarized M0φsor Pi-activated MPiφs. (<b>A, B</b>) Extracellular ATP and PPi released by the indicated macrophage types. (<b>C</b>) PPi/ATP ratio. Statistical significance was determined by unpaired Student’s <i>t</i>-test. Results are presented as mean ± SE of 3 independent experiments with 3 mice per experiment. (<b>D</b>) Calcium deposition on fixed VSMCs in absence (Control) or presence of M0φs or MPiφs. Experiments were performed in the presence or absence of exogenous alkaline phosphatase (-AP and +AP). Statistical significance was determined by one-way ANOVA analysis of variance followed by Tukey’s multicomparison test. Results are presented as mean ± SE of 4 independent experiments with 3 mice per experiment. *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001.</p

MPiφs have an enhanced energetic profile.

<p>(<b>A</b>) RNAseq data summarizing fold mRNA downregulation or upregulation in MPiφs of enzymes in the main pathways involved in glucose and fatty acid catabolism. (<b>B</b>) mRNA levels for <i>HIF-1α</i> and <i>PGC-1β</i> mRNA levels. (<b>C</b>) Intracellular ATP and ADP/ATP ratio. Statistical significance was determined by Student’s <i>t</i>-test. Results are presented as mean ± SE of 4 independent experiments with 3 mice per experiment. **, <i>P</i><0.01; ***, <i>P</i><0.001.</p

MPiφs show elevated antioxidant synthesis.

<p>(<b>A</b>) RNAseq data summarizing fold mRNA downregulation or upregulation in MPiφs of enzymes involved in glutathione metabolism. (<b>B</b>) Total antioxidant capacity. (<b>C</b>) GSH/GSSG ratio. (<b>D</b>) Total GSH. Statistical significance was determined Student’s <i>t</i>-test. Results are presented as mean ± SE of 4 independent experiments with 3 mice per experiment. *, <i>P</i><0.05; **, <i>P</i><0.01.</p

MPiφs degrade arginine via Arg1.

<p>(<b>A</b>) RNAseq data summarizing fold mRNA upregulation in MPiφs of collagen types and enzymes involved in the urea cycle. (<b>B</b>) <i>Arg1</i> and <i>iNOS</i> mRNA levels. (<b>C</b>) Arginase activity. Statistical significance was determined by Student’s <i>t</i>-test. Results are presented as mean ± SE of 4 independent experiments with 3 mice per experiment. ***, <i>P</i><0.001.</p

Macrophages are activated by high Pi concentration.

<p>Unpolarized M0φs were washed three times and incubated with the indicated concentration of Pi for 3 days (<b>A</b>) or incubated with 2.5 mmol/L Pi for the indicated time (<b>B</b>). Statistical significance was determined by one-way ANOVA analysis of variance followed by Tukey´s multicomparison test. (<b>C</b>) Flow cytometry studies showing activation of M0φs after exposure to high [Pi] (2.5 mmol/L) for 7 days. (<b>D</b>) Quantification of CD11b and F4/80, revealing significantly higher levels of macrophage-specific surface markers on MPiφs than on M0φs. Statistical significance was determined by Student’s <i>t</i>-test. Results are presented as mean ± SE of 3 independent experiments with 3 mice per experiment.*, <i>P</i><0.05; **,<i>P</i><0.01; ***,<i>P</i><0.001.</p

Deletion of miR-146a gene in hematopoietic cells does not increase adhesion in <i>Ldlr</i>^-/- mice fed HFD.

<p>Lethally-irradiated <i>Ldlr</i>^<i>-/-</i> mice (CD45.1, 8 to 10-week-old) were transplanted with cells obtained from bone marrow (BM) of wt or <i>miR-146a</i>^-/- mice (both CD45.2). After 4 weeks of recovery, mice were challenged with a high-fat diet (HFD) for 20 weeks and analyzed by intravital microscopy in the cremaster muscle. Images are representative for Ly6C⁺ monocytes, Ly6G⁺ neutrophils, and CD4⁺ T cells adhered to the wall of cremasteric venules. Dashed lines mark the boundaries of the venules.</p

Ablation of miR-146a gene in hematopoietic cells does not affect diet-induced atherosclerosis in <i>Ldlr</i>^-/- mice.

<p>Lethally-irradiated <i>Ldlr</i>^<i>-/-</i> mice (CD45.1, 8 to 10-week-old) were transplanted with cells obtained from bone marrow (BM) of wt or <i>miR-146a</i>^-/- mice (both CD45.2). After 4 weeks of recovery, mice were challenged with a high-fat diet (HFD). Mice were euthanized after 8 or 20 weeks of HFD and tissues were harvested for immunohistopathological characterization of atherosclerotic lesions. Representative images are shown in each panel. <b>(A)</b> Percentage of aortic arch are occupied by atheroma visualized by <i>en face</i> Oil Red O staining. <b>(B)</b> Quantification of atheroma size in hematoxylin/eosin-stained sections of the aortic sinus. <b>(C)</b> Area of atherosclerotic lesions occupied by necrotic cores quantified in histological sections from the aortic sinus. *: p<0.05, ***: p<0.001 vs same BM genotype at 20 weeks of HFD.</p

Study design and efficiency of bone marrow transplantation.

<p><b>(A)</b> Lethally-irradiated <i>Ldlr</i>^<i>-/-</i> mice (CD45.1, 8 to 10-week-old) were transplanted with cells obtained from bone marrow (BM) of wt or <i>miR-146a</i>^-/- mice (both CD45.2). After 4 weeks of recovery, mice were challenged with a high-fat diet for 8 and 20 weeks. Blood and aorta were collected for biochemical and expression studies and to quantify atherosclerosis burden. <b>(B)</b> Quantification of transplant efficiency in both groups of mice as determined by flow cytometry of blood extracted 1 month after BM transplant. Donor and host cells are CD45.2- and CD45.1-immunoreactive, respectively. Representative flow cytometry profiles are shown below the chart. <b>(C)</b> Body weight at different times after the onset of HFD. <b>(D)</b> Pre and post diet miR-146a levels in blood leukocytes were measured by qRT-PCR. The 2^-ΔCt method was used to calculate miRNA abundance relative to U6 (Ct = Threshold Cycle; ΔCt = Ct sample gene − Ct endogenous control). **: p<0.01, ***: p<0.001 BM wt vs BM <i>miR-146a</i>^-/- at both time points. (<b>E</b>) Spleen from BM wt or BM <i>miR-146a</i>^-/- mice were weighed after 20 weeks of HFD. Shown are representative images of spleens and femurs.</p