p600 Plays Essential Roles in Fetal Development

p600 is a multifunctional protein implicated in cytoskeletal organization, integrin-mediated survival signaling, calcium-calmodulin signaling and the N-end rule pathway of ubiquitin-proteasome-mediated proteolysis. While push, the Drosophila counterpart of p600, is dispensable for development up to adult stage, the role of p600 has not been studied during mouse development. Here we generated p600 knockout mice to investigate the in vivo functions of p600. Interestingly, we found that homozygous deletion of p600 results in lethality between embryonic days 11.5 and 13.5 with severe defects in both embryo and placenta. Since p600 is required for placental development, we performed conditional disruption of p600, which deletes selectively p600 in the embryo but not in the placenta. The conditional mutant embryos survive longer than knockout embryos but ultimately die before embryonic day 14.5. The mutant embryos display severe cardiac problems characterized by ventricular septal defects and thin ventricular walls. These anomalies are associated with reduced activation of FAK and decreased expression of MEF2, which is regulated by FAK and plays a crucial role in cardiac development. Moreover, we observed pleiotropic defects in the liver and brain. In sum, our study sheds light on the essential roles of p600 in fetal development

Application of thermosensitive-hydrogel combined with dental pulp stem cells on the injured fallopian tube mucosa in an animal model

Objectives: Fallopian tube (FT) injury is an important factor that can lead to tubal infertility. Stem-cell-based therapy shows great potential for the treatment of injured fallopian tube. However, little research has shown that mesenchymal stem cells (MSCs) can be used to treat fallopian tube damage by in situ injection. In this study, we in situ transplanted PF127 hydrogel encapsulating dental pulp stem cells (DPSCs) into the injured sites to promote the repair and regeneration of fallopian tube injury.Materials and methods: The properties of dental pulp stem cells were evaluated by flow cytometry, immunofluorescence analysis, and multi-differentiation detection. The immunomodulatory and angiogenic characteristics of dental pulp stem cells were analyzed on the basis of the detection of inflammatory factor expression and the formation of capillary-like structures, respectively. The biocompatibility of PF127 hydrogel was evaluated by using Live/Dead and CCK-8 assays. The effects of PF127 hydrogel containing dental pulp stem cells on the repair and regeneration of fallopian tube injury were evaluated by histological analysis [e.g., hematoxylin and eosin (H&E) and Masson’s trichrome staining, TUNEL staining, immunofluorescence staining, and immunohistochemistry], Enzyme-linked immunosorbent assay (ELISA), and RT-PCR detections.Results: Dental pulp stem cells had MSC-like characteristics and great immunomodulatory and angiogenic properties. PF127 hydrogel had a thermosensitive feature and great cytocompatibility with dental pulp stem cells. In addition, our results indicated that PF127 hydrogel containing dental pulp stem cells could promote the repair and regeneration of fallopian tube damage by inhibiting cell apoptosis, stimulating the secretion of angiogenic factors, promoting cell proliferation, modulating the secretion of inflammatory factors, and restoring the secretion of epithelial cells.Conclusion: In this study, our results reported that in situ injection of PF127 hydrogel encapsulating dental pulp stem cells into the injured sites could provide an attractive strategy for the future treatment of fallopian tube injury in clinical settings

název v českém jazyce není uveden

1. lékařská fakultaFirst Faculty of Medicin

Deletion of the p27(Kip1) gene restores normal development in cyclin D1-deficient mice

D-type cyclins (cyclins D1, D2, and D3) are key components of cell cycle machinery in mammalian cells. These proteins are believed to drive cell cycle progression by associating with their kinase partners, cyclin-dependent kinases, and by directing phosphorylation of critical cellular substrates. In addition, D-cyclins play a kinase-independent role by sequestering cell cycle inhibitors p27(Kip1) and p21(Cip1). In the past, we and others generated cyclin D1-deficient mice and have shown that these mice display developmental abnormalities, hypoplastic retinas, and pregnancy-insensitive mammary glands. To test the significance of cyclin D1–p27(Kip1) interaction within a living mouse, we crossed cyclin D1-deficient mice with mice lacking p27(Kip1), and we generated double-mutant cyclin D1(−/−)p27(−/−) animals. Here we report that ablation of p27(Kip1) restores essentially normal development in cyclin D1-deficient mice. Our results provide genetic evidence that p27(Kip1) functions downstream of cyclin D1

Essential Role for Cyclin D3 in Granulocyte Colony-Stimulating Factor-Driven Expansion of Neutrophil Granulocytes

The proliferation of neutrophil granulocyte lineage is driven largely by granulocyte colony-stimulating factor (G-CSF) acting via the G-CSF receptors. In this study, we show that mice lacking cyclin D3, a component of the core cell cycle machinery, are refractory to stimulation by the G-CSF. Consequently, cyclin D3-null mice display deficient maturation of granulocytes in the bone marrow and have reduced levels of neutrophil granulocytes in their peripheral blood. The mutant mice are unable to mount a normal response to bacterial challenge and succumb to microbial infections. In contrast, the expansion of hematopoietic stem cells and lineage-committed myeloid progenitors proceeds relatively normally in mice lacking cyclin D3, revealing that the requirement for cyclin D3 function operates at later stages of neutrophil development. Importantly, we verified that this requirement is specific to cyclin D3, as mice lacking other G(1) cyclins (D1, D2, E1, or E2) display normal granulocyte counts. Our analyses revealed that in the bone marrow cells of wild-type mice, activation of the G-CSF receptor leads to upregulation of cyclin D3. Collectively, these results demonstrate that cyclin D3 is an essential cell cycle recipient of G-CSF signaling, and they provide a molecular link of how G-CSF-dependent signaling triggers cell proliferation

Defects in FAK activation in conditional <i>p600</i> knockout heart.

<p>Transverse sections of <i>p600</i>^−/+ (<i>p600</i>^+/−/<i>Sox2-Cre</i>⁺) (left) and <i>p600</i> cKO (right) heart at days E13.5 were stained with anti-desmin (A), anti-FAK (B), anti-phospho-FAK (Tyr397) (C), and anti-MEF2 (D) antibodies. In panels C and D, the position of the inferior atrioventricular endocardial cushions (IC) is labeled. Scale bars indicate 200 µm.</p

Conditional <i>p600</i> knockout specific to embryos.

<p>(A) Conditional deletion strategy of <i>p600</i> allele in embryos. The mice with ‘Neo allele’ were mated with transgenic mice which expresses <i>actin</i> promoter-driven <i>flippase</i>. This crossing eliminated the Neo cassette region between the frt sites (white rectangles), resulting in ‘floxed allele’. After crossing with Tg(<i>Sox2-Cre</i>) mice <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066269#pone.0066269-Hayashi1" target="_blank">[21]</a>, the region between loxP sites (red triangles) including the first exon of <i>p600</i> is deleted where Cre recombinase is expressed, resulting in ‘KO allele’. (B) Genotype analysis of <i>Sox2-Cre</i>-mediated p600 knockout animals. Male mice of Tg (<i>Sox2-Cre</i>) <i>p600</i>^+/− were crossed with female containing the homozygous <i>p600</i> floxed alleles (<i>p600</i>^flox/flox). Genotypes of resulting littermates were analyzed by PCR. The <i>p</i>-values of X² test are shown. * and ** show significant differences between observed frequency of survival <i>p600</i> cKO embryos and expected frequency of 25%, according to Mendelian distribution of the genotypes with p-score <0.05 and <0.01, respectively. (C) Typical appearances of <i>p600</i> cKO and heterozygous <i>p600</i>^+/− (<i>p600</i>^+/−/<i>Sox2 Cre</i>⁺) embryos at days E12.5 and E13.5. Scale bars indicate 1 mm.</p