Nox4-IGF2 axis promotes differentiation of embryoid body cells into derivatives of the three embryonic germ layers
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ABSTRACT: Reactive oxygen species (ROS) play important roles as second messengers in a wide array of cellular processes including differentiation of stem cells. We identified Nox4 as the major ROS generating enzyme whose expression is induced during differentiation of embryoid body (EB) into cells of all three germ layers. The role of Nox4 was examined using iPS cells generated from Nox4 -/- mouse. Differentiation markers showed significantly reduced expression levels consistent with the importance of Nox4-generated ROS during this process. From transcriptomic analyses, we found insulin-like growth factor 2 (IGF2), a member of a gene family extensively involved in embryonic development, as one of the most down-regulated genes. Indeed, addition of IGF2 to culture partly restored the differentiation efficiency of Nox4-/- iPS cells. Our results reveal an important signaling axis mediated by ROS in control of crucial events during differentiation of pluripotent stem cells.
Project description:Tightly regulated and cell-specific NADPH-oxidases (Nox) represent one of the major sources of reactive oxygen species (ROS) signaling molecules that are involved in tissue development and stem cell self-renewal. We have characterized the role of Nox4 in osteo-progenitors during postnatal bone development. Nox4 expression in bone and ROS generation were increased during early osteoblast differentiation and bone development. Stromal osteoblastic cell self-renewal, proliferation and ROS production were significantly lower in samples from whole body Nox4 knockout mice (Nox4-/-) and conditional knockout (CKO) mice with depletion of Nox4 in the limb bud mesenchyme compared with those from control mice (Nox4fl/fl), but they were reversed after 9 passages. In both sexes, bone volume, trabecular number and bone mineral density were significantly lower in 3-week old CKO and Nox4-/- mice compared with Nox4fl/fl controls. This was reflected in serum levels of bone formation markers alkaline phosphatase (ALP) and procollagen 1 intact N-terminal propeptide (P1NP). However, under-developed bone formation in 3-week old CKO and Nox4-/- mice quickly caught up to levels of control mice by 6-week of age, remained no different at 13-week of age, and was reversed in 32-week old male mice. Osteoclastogenesis showed no differences among groups, however, CTX1 reflecting osteoclast activity was significantly higher in 3-week old male CKO and Nox4-/- mice compared with control mice, and significantly lower in 32-week old Nox4-/- mice compared with control mice. These data suggest that Nox4 expression and ROS signaling in bone and osteoblastic cells coordinately play a role on osteoblast differentiation, proliferation and maturation.
Project description:Differentiation of fibroblasts to myofibroblasts is governed by the transforming growth factor beta (TGF-β) through a mechanism involving redox signaling and generation of reactive oxygen species (ROS). Myofibroblasts synthesize proteins of the extracellular matrix and display a contractile phenotype. Myofibroblasts are predominant contributors of wound healing and several pathological states, including fibrotic diseases and cancer. Inhibition of the ROS-generating enzyme NADPH oxidase 4 (NOX4) has been proposed to mitigate fibroblast to myofibroblast differentiation and to offer a therapeutic option for the treatment of fibrotic diseases. In this study, we addressed the role of NOX4 in physiological wound healing and in TGF-β-induced myofibroblast differentiation. We explored the phenotypic changes induced by TGF-β in primary skin fibroblasts isolated from Nox4-deficient mice by immunofluorescence, Western blotting and RNA sequencing. Mice deficient for Cyba, the gene coding for p22phox, a key subunit of NOX4 were used for confirmatory experiments as well as human primary skin fibroblasts. In vivo, the wound healing was similar in wild-type and Nox4-deficient mice. In vitro, despite a strong upregulation following TGF-β treatment, Nox4 did not influence skin myofibroblast differentiation. Nevertheless, up-regulation of the mitochondrial protein Ucp2 and the stress-response protein Hddc3, as well as down-regulation of Islr were observed in Nox4-deficient fibroblasts. Altogether, we provide extensive evidence challenging a profibrotic role of NOX4 in skin fibroblasts and show that Nox4 regulates Ucp2 and Hddc3 expression, suggesting the presence of a so far undescribed redox crosstalk between NOX4 and redox homeostasis in fibroblasts.
Project description:Hypoxic stress is a feature of rapidly growing thyroid tumors. Cancer progression is thought to be driven by reactive oxygen species (ROS) induced hypoxia adaptation. NADPH oxidases (NOXs), which produce ROS as their primary and sole function, has become of particular interest in thyroid malignancy. NOX4 was demonstrated to be upregulated in papillary thyroid cancers, functioning as a mitochondrial energetic sensor to modulate ATP levels, mediating overproduction of ROS induced by IL-δ, inversely correlating to thyroid differentiation. In this study, we analyzed hypoxia-treated BCPAP cells transfected with siRNA against NOX4. Results provides insight into the role of NOX4 in hypoxia adaptation.
Project description:NADPH oxidases are the only know enzyme family that has reactive oxygen species (ROS, e.g. superoxide and hydrogen peroxide) as their main metabolic product. They are therefore a prima candidate gene family to define a molecular source for ROS in physiology and pathophysiology. The type 4 NADPH oxidase (NOX4) is the most abundant isoform with the highest basal expression levels.The Nox4 knockout mouse was designed using a cre/lox recombination technique which allows to create either constitutive (type III recombination) and conditional (type I recombination) knockout alleles.
Project description:NADPH oxidases are the only know enzyme family that has reactive oxygen species (ROS, e.g. superoxide and hydrogen peroxide) as their main metabolic product. They are therefore a prima candidate gene family to define a molecular source for ROS in physiology and pathophysiology. The type 4 NADPH oxidase (NOX4) is the most abundant isoform with the highest basal expression levels.The Nox4 knockout mouse was designed using a cre/lox recombination technique which allows to create either constitutive (type III recombination) and conditional (type I recombination) knockout alleles. cDNA microarray experiments of brain, kidney, muscle and pancreas of four male mutant mice versus a RNA pool of four male wild type mice. For each animal two technical replicates were performed including a dye swap experiment. All animals with the same age of 17 weeks on a C57BL/6 background.
Project description:Periodontitis and infections with periodontal bacteria have been highlighted as risk factors for dementia. In recent years attention has been drawn to the role of microglia cells in neurodegenerative diseases. However, there is limited knowledge of the influence of periodontal bacteria on microglia cells. The aim of the present study was to investigate the interactions between the periodontal bacteria P. gingivalis and microglia cells, and to unravel if these interactions could contribute to Alzheimer’s pathology. Periodontitis and infections with periodontal bacteria have been highlighted as risk factors for dementia. In recent years attention has been drawn to the role of microglia cells in neurodegenerative diseases. However, there is limited knowledge of the influence of periodontal bacteria on microglia cells. The aim of the present study was to investigate the interactions between the periodontal bacteria P. gingivalis and microglia cells, and to unravel if these interactions could contribute to Alzheimer’s pathology. We found, through microarray analysis, that stimulation of microglia cells with P. gingivalis resulted in upregulation of several Alzheimer´s disease associated genes, including NOX4. We also showed that P. gingivalis LPS mediated ROS production and IL-6 and IL-8 induction via NOX4 in microglia. The viability of neurons was shown to be reduced by conditioned media from microglia cells stimulated with P. gingivalis LPS, and that the reduction was NOX4 dependent. The levels of total and phosphorylated Tau in neurons were increased by conditioned media from microglia cells stimulated with P. gingivalis or LPS. This increase was NOX4 dependent. In summary our findings give us a potential mechanistic explanation of how the periodontal pathogen P. gingivalis could trigger or exacerbate AD pathogenesis.
Project description:Within the family of NADPH oxidases, Nox4 is unique as it is predominantly localized in the endoplasmic reticulum, has constitutive activity and generates H2O2. We hypothesize that these features are consequences of a so far unidentified Nox4-interacting protein. Interacting proteins were screened by quantitative SILAC-Co-immunoprecipitation in HEK293 cells stably overexpressing Nox4. By this technique, several interacting proteins were identified with calnexin showing the most robust interaction.
Project description:Gene expression profiling of mouse articular chondrocytes infected with Ad-C or Ad-Nox4 In this study, we have attempted to explore the effects of Nox4 and have identified numerous genes which are involved in osteoarthritis pathogenesis.