The chronic skin inflammation psoriasis is crucially dependent on the IL-23/IL-17 cytokine axis. Although IL-23 is expressed by psoriatic keratinocytes and immune cells, only the immune cell-derived ...IL-23 is believed to be disease relevant. Here we use a genetic mouse model to show that keratinocyte-produced IL-23 is sufficient to cause a chronic skin inflammation with an IL-17 profile. Furthermore, we reveal a cell-autonomous nuclear function for the actin polymerizing molecule N-WASP, which controls IL-23 expression in keratinocytes by regulating the degradation of the histone methyltransferases G9a and GLP, and H3K9 dimethylation of the IL-23 promoter. This mechanism mediates the induction of IL-23 by TNF, a known inducer of IL-23 in psoriasis. Finally, in keratinocytes of psoriatic lesions a decrease in H3K9 dimethylation correlates with increased IL-23 expression, suggesting relevance for disease. Taken together, our data describe a molecular pathway where epigenetic regulation of keratinocytes can contribute to chronic skin inflammation.
A diverse set of cellular defects, presumably elicited by multiple genetic alterations, underlies cancer development. Aberrant Hedgehog (Hh) signaling has recently been implicated in the development ...and maintenance of breast cancer. However, evidence conclusively showing that activated Hh signaling can induce mammary tumors is lacking. We now show that transgenic expression of the Hh effector protein GLI1 under the regulation of the mouse mammary tumor virus promoter, expressed in the mouse mammary gland, is associated with the appearance of hyperplastic lesions, defective terminal end buds, and tumor development. The GLI1-induced tumors are histologically heterogeneous and involve the expansion of a population of epithelial cells expressing the progenitor cell markers keratin 6 and Bmi-1. Moreover, tumor cells express genes involved in proliferation, cell survival, and metastasis. GLI1-induced tumors do not fully regress following transgene deinduction, indicating that some tumors develop and are maintained autonomously, independent of sustained transgenic GLI1 expression. The data strongly support a role of Hh/GLI signaling in breast cancer development and suggest that inhibition of this signaling pathway represents a new therapeutic opportunity for limiting tumorigenesis and early tumorigenic progression.
Ro52/Trim21 is targeted as an autoantigen in systemic lupus erythematosus and Sjögren's syndrome. Polymorphisms in the Ro52 gene have been linked to these autoimmune conditions, but the molecular ...mechanism by which Ro52 may promote development of systemic autoimmune diseases has not been explored. To address this issue, we generated Ro52-null mice (Ro52(-/-)), which appear phenotypically normal if left unmanipulated. However, Ro52(-/-) mice develop severe dermatitis extending from the site of tissue injury induced by ear tags. The affected mice further develop several signs of systemic lupus with hypergammaglobulinemia, autoantibodies to DNA, proteinuria, and kidney pathology. Ro52, which was recently identified as an E3 ligase, mediates ubiquitination of several members of the interferon regulatory factor (IRF) family, and the Ro52-deficient mice have an enhanced production of proinflammatory cytokines that are regulated by the IRF transcription factors, including cytokines involved in the Th17 pathway (interleukin IL 6, IL-12/IL-23p40, and IL-17). Loss of IL-23/IL-17 by genetic deletion of IL-23/p19 in the Ro52(-/-) mice conferred protection from skin disease and systemic autoimmunity. These data reveal that the lupus-associated Ro52 protein is an important negative regulator of proinflammatory cytokine production, and they provide a mechanism by which a defective Ro52 function can lead to tissue inflammation and systemic autoimmunity through the IL-23-Th17 pathway.
Abstract Neuronal disorders, like Huntington's disease (HD), are difficult to study, due to limited cell accessibility, late onset manifestations, and low availability of material. The establishment ...of an in vitro model that recapitulates features of the disease may help understanding the cellular and molecular events that trigger disease manifestations. Here, we describe the generation and characterization of a series of induced pluripotent stem (iPS) cells derived from patients with HD, including two rare homozygous genotypes and one heterozygous genotype. We used lentiviral technology to transfer key genes for inducing reprogramming. To confirm pluripotency and differentiation of iPS cells, we used PCR amplification and immunocytochemistry to measure the expression of marker genes in embryoid bodies and neurons. We also analyzed teratomas that formed in iPS cell-injected mice. We found that the length of the pathological CAG repeat did not increase during reprogramming, after long term growth in vitro, and after differentiation into neurons. In addition, we observed no differences between normal and mutant genotypes in reprogramming, growth rate, caspase activation or neuronal differentiation. However, we observed a significant increase in lysosomal activity in HD-iPS cells compared to control iPS cells, both during self-renewal and in iPS-derived neurons. In conclusion, we have established stable HD-iPS cell lines that can be used for investigating disease mechanisms that underlie HD. The CAG stability and lysosomal activity represent novel observations in HD-iPS cells. In the future, these cells may provide the basis for a powerful platform for drug screening and target identification in HD.
Retro-bulbar sinus puncture and facial vein phlebotomy are two widely used methods for blood sampling in laboratory mice. However, the animal welfare implications associated with these techniques are ...currently debated, and the possible physiological and pathological implications of blood sampling using these methods have been sparsely investigated. Therefore, this study was conducted to assess and compare the impacts of blood sampling by retro-bulbar sinus puncture and facial vein phlebotomy. Blood was obtained from either the retro-bulbar sinus or the facial vein from male C57BL/6J mice at two time points, and the samples were analyzed for plasma corticosterone. Body weights were measured at the day of blood sampling and the day after blood sampling, and the food consumption was recorded automatically during the 24 hours post-procedure. At the end of study, cheeks and orbital regions were collected for histopathological analysis to assess the degree of tissue trauma. Mice subjected to facial vein phlebotomy had significantly elevated plasma corticosterone levels at both time points in contrast to mice subjected to retro-bulbar sinus puncture, which did not. Both groups of sampled mice lost weight following blood sampling, but the body weight loss was higher in mice subjected to facial vein phlebotomy. The food consumption was not significantly different between the two groups. At gross necropsy, subcutaneous hematomas were found in both groups and the histopathological analyses revealed extensive tissue trauma after both facial vein phlebotomy and retro-bulbar sinus puncture. This study demonstrates that both blood sampling methods have a considerable impact on the animals' physiological condition, which should be considered whenever blood samples are obtained.
The Hedgehog (Hh) pathway plays important roles during embryogenesis and carcinogenesis. Here, we show that ablation of the mouse Suppressor of fused (
Sufu), an intracellular pathway component, ...leads to embryonic lethality at ∼E9.5 with cephalic and neural tube defects. Fibroblasts derived from
Sufu
−/−
embryos showed high Gli-mediated Hh pathway activity that could not be modulated at the level of Smoothened and could only partially be blocked by PKA activation. Despite the robust constitutive pathway activation in the
Sufu
−/−
fibroblasts, the GLI1 steady-state localization remained largely cytoplasmic, implying the presence of an effective nuclear export mechanism.
Sufu
+/−
mice develop a skin phenotype with basaloid changes and jaw keratocysts, characteristic features of Gorlin syndrome, a human genetic disease linked to enhanced Hh signaling. Our data demonstrate that, in striking contrast to
Drosophila, in mammals, Sufu has a central role, and its loss of function leads to potent ligand-independent activation of the Hh pathway.
Tetradecylthioacetic acid (TTA) is a synthetic fatty acid with a sulfur substitution in the β-position. This modification renders TTA unable to undergo complete β-oxidation and increases its ...biological activity, including activation of peroxisome proliferator activated receptors (PPARs) with preference for PPARα. This study investigated the effects of TTA on lipid and lipoprotein metabolism in the intestine and liver of mice fed a high fat diet (HFD). Mice receiving HFD supplemented with 0.75% (w/w) TTA had significantly lower body weights compared to mice fed the diet without TTA. Plasma triacylglycerol (TAG) was reduced 3-fold with TTA treatment, concurrent with increase in liver TAG. Total cholesterol was unchanged in plasma and liver. However, TTA promoted a shift in the plasma lipoprotein fractions with an increase in larger HDL particles. Histological analysis of the small intestine revealed a reduced size of lipid droplets in enterocytes of TTA treated mice, accompanied by increased mRNA expression of fatty acid transporter genes. Expression of the cholesterol efflux pump Abca1 was induced in the small intestine, but not in the liver. Scd1 displayed markedly increased mRNA and protein expression in the intestine of the TTA group. It is concluded that TTA treatment of HFD fed mice leads to increased expression of genes involved in uptake and transport of fatty acids and HDL cholesterol in the small intestine with concomitant changes in the plasma profile of smaller lipoproteins.
The growth of stem cells in in vitro conditions requires optimal balance between signals mediating cell survival, proliferation, and self-renewal. For clinical application of stem cells, the use of ...completely defined conditions and elimination of all animal-derived materials from the establishment, culture, and differentiation processes is desirable.
Here, we report the development of a fully defined xeno-free medium (RegES), capable of supporting the expansion of human embryonic stem cells (hESC), induced pluripotent stem cells (iPSC) and adipose stem cells (ASC). We describe the use of the xeno-free medium in the derivation and long-term (>80 passages) culture of three pluripotent karyotypically normal hESC lines: Regea 06/015, Regea 07/046, and Regea 08/013. Cardiomyocytes and neural cells differentiated from these cells exhibit features characteristic to these cell types. The same formulation of the xeno-free medium is capable of supporting the undifferentiated growth of iPSCs on human feeder cells. The characteristics of the pluripotent hESC and iPSC lines are comparable to lines derived and cultured in standard undefined culture conditions. In the culture of ASCs, the xeno-free medium provided significantly higher proliferation rates than ASCs cultured in medium containing allogeneic human serum (HS), while maintaining the differentiation potential and characteristic surface marker expression profile of ASCs, although significant differences in the surface marker expression of ASCs cultured in HS and RegES media were revealed.
Our results demonstrate that human ESCs, iPSCs and ASCs can be maintained in the same defined xeno-free medium formulation for a prolonged period of time while maintaining their characteristics, demonstrating the applicability of the simplified xeno-free medium formulation for the production of clinical-grade stem cells. The basic xeno-free formulation described herein has the potential to be further optimized for specific applications relating to establishment, expansion and differentiation of various stem cell types.
Hutchinson-Gilford progeria syndrome (HGPS) is a very rare genetic disorder that is characterized by multiple features of premature aging and largely affects tissues of mesenchymal origin. In this ...study, we describe the development of a tissue-specific mouse model that overexpresses the most common HGPS mutation (LMNA, c.1824C>T, p.G608G) in osteoblasts. Already at the age of 5 weeks, HGPS mutant mice show growth retardation, imbalanced gait and spontaneous fractures. Histopathological examination revealed an irregular bone structure, characterized by widespread loss of osteocytes, defects in mineralization, and a hypocellular red bone marrow. Computerized tomography analysis demonstrated impaired skeletal geometry and altered bone structure. The skeletal defects, which resemble the clinical features reported for bone disease in HGPS patients, was associated with an abnormal osteoblast differentiation. The osteoblast-specific expression of the HGPS mutation increased DNA damage and affected Wnt signaling. In the teeth, irregular dentin formation, as was previously demonstrated in human progeria cases, caused severe dental abnormalities affecting the incisors. The observed phenotype also shows similarities to reported bone abnormalities in aging mice and may therefore help to uncover general principles of the aging process.
Background: Tissue-specific mouse model for Hutchinson-Gilford progeria syndrome (HGPS).
Results: Dysfunctional osteoblast maturation and impacts DNA damage and Wnt signaling, which lead to abnormal bone mineralization and impaired skeletal and dental structures.
Conclusion: Resemble clinical features of HGPS and normal bone aging.
Significance: Provides insights to the molecular mechanisms of HGPS and will be useful to further elucidate the processes that contribute to general bone aging.
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