The epigenetic mechanism of mechanically induced osteogenic differentiation

Epigenetic regulation of gene expression occurs due to alterations in chromatin proteins that do not change DNA sequence, but alter the chromatin architecture and the accessibility of genes, resulting in changes to gene expression that are preserved during cell division. Through this process genes are switched on or off in a more durable fashion than other transient mechanisms of gene regulation, such as transcription factors. Thus, epigenetics is central to cellular differentiation and stem cell linage commitment. One such mechanism is DNA methylation, which is associated with gene silencing and is involved in a cell’s progression towards a specific fate. Mechanical signals are a crucial regulator of stem cell behavior and important in tissue differentiation; however, there has been no demonstration of a mechanism whereby mechanics can affect gene regulation at the epigenetic level. In this study, we identified candidate DNA methylation sites in the promoter regions of three osteogenic genes from bone marrow derived mesenchymal stem cells (MSCs). We demonstrate that mechanical stimulation alters their epigenetic state by reducing DNA methylation and show an associated increase in expression. We contrast these results with biochemically induced differentiation and distinguish expression changes associated with durable epigenetic regulation from those likely to be due to transient changes in regulation. This is an important advance in stem cell mechanobiology as it is the first demonstration of a mechanism by which the mechanical micro-environment is able to induce epigenetic changes that control osteogenic cell fate, and that can be passed to daughter cells. This is a first step to understanding that will be vital to successful bone tissue engineering and regenerative medicine, where continued expression of a desired long-term phenotype is crucial

Dichloroacetate prevents cisplatin-induced nephrotoxicity without compromising cisplatin anticancer properties

Cisplatin is an effective anticancer drug; however, cisplatin use often leads to nephrotoxicity, which limits its clinical effectiveness. In this study, we determined the effect of dichloroacetate, a novel anticancer agent, in a mouse model of cisplatin-induced AKI. Pretreatment with dichloroacetate significantly attenuated the cisplatin-induced increase in BUN and serum creatinine levels, renal tubular apoptosis, and oxidative stress. Additionally, pretreatment with dichloroacetate accelerated tubular regeneration after cisplatin-induced renal damage. Whole transcriptome sequencing revealed that dichloroacetate prevented mitochondrial dysfunction and preserved the energy-generating capacity of the kidneys by preventing the cisplatin-induced downregulation of fatty acid and glucose oxidation, and of genes involved in the Krebs cycle and oxidative phosphorylation. Notably, dichloroacetate did not interfere with the anticancer activity of cisplatin in vivo. These data provide strong evidence that dichloroacetate preserves renal function when used in conjunction with cisplatin

Glutathione Transferase Omega-1 Regulates NLRP3 Inflammasome Activation through NEK7 Deglutathionylation

The NLRP3 inflammasome is a cytosolic complex sensing phagocytosed material and various damage-associated molecular patterns, triggering production of the pro-inflammatory cytokines interleukin-1 beta (IL)-1β and IL-18 and promoting pyroptosis. Here, we characterize glutathione transferase omega 1-1 (GSTO1-1), a constitutive deglutathionylating enzyme, as a regulator of the NLRP3 inflammasome. Using a small molecule inhibitor of GSTO1-1 termed C1-27, endogenous GSTO1-1 knockdown, and GSTO1-1−/− mice, we report that GSTO1-1 is involved in NLRP3 inflammasome activation. Mechanistically, GSTO1-1 deglutathionylates cysteine 253 in NIMA related kinase 7 (NEK7) to promote NLRP3 activation. We therefore identify GSTO1-1 as an NLRP3 inflammasome regulator, which has potential as a drug target to limit NLRP3-mediated inflammation.We would like to acknowledge the following grants: the National Health and Medical Research Council of Australia (NHMRC) is thanked for Project Grant APP1124673 to P.G.B., M.G.C., and L.A.J.O.; Principal Research Fellowship 1117602 to J.B.B.; and NHMRC Project Grant APP1156455 to J.B.B., P.G.B., and M.G.C. The O’Neill laboratory acknowledges the following grant support: European Research Council (ECFP7-ERC-MICROINNATE) and Science Foundation Ireland Investigator Award (SFI 12/IA/1531)

Helicobacter pylori induces somatic mutations in TP53 via overexpression of CHAC1 in infected gastric epithelial cells

Infection with Helicobacter pylori is known to decrease the level of glutathione in gastric epithelial cells and increase the production of reactive oxygen species (ROS), which can lead to DNA damage and the development of gastric cancer. Cation transport regulator 1 (CHAC1) has γ-glutamylcyclotransferase activity that degrades glutathione. We found that cagA-positive H. pylori infection triggered CHAC1 overexpression in human gastric epithelial (AGS) cells leading to glutathione degradation and the accumulation of ROS. Nucleotide alterations in the TP53 tumour suppressor gene were induced in AGS cells overexpressing CHAC1, whereas no mutations were detected in cells overexpressing a catalytically inactive mutant of CHAC1. A high frequency of TP53 mutations occurred in H. pylori-infected AGS cells, but this was prevented in cells transfected with CHAC1 siRNA. These findings indicate that H. pylori-mediated CHAC1 overexpression degrades intracellular glutathione, allowing the accumulation of ROS which subsequently causes mutations that could contribute to the development of gastric cancer.This work was supported by the Japan Society for the Promotion of Science KAKENHI (16K19077) and Project Grant 525458 from the Australian National Health and Medical Research Council

GSTO1-1 plays a pro-inflammatory role in models of inflammation, colitis and obesity

Glutathione transferase Omega 1 (GSTO1-1) is an atypical GST reported to play a pro-inflammatory role in response to LPS. Here we show that genetic knockout of Gsto1 alters the response of mice to three distinct inflammatory disease models. GSTO1-1 deficiency ameliorates the inflammatory response stimulated by LPS and attenuates the inflammatory impact of a high fat diet on glucose tolerance and insulin resistance. In contrast, GSTO1-1 deficient mice show a more severe inflammatory response and increased escape of bacteria from the colon into the lymphatic system in a dextran sodium sulfate mediated model of inflammatory bowel disease. These responses are similar to those of TLR4 and MyD88 deficient mice in these models and confirm that GSTO1-1 is critical for a TLR4-like pro-inflammatory response in vivo. In wild-type mice, we show that a small molecule inhibitor that covalently binds in the active site of GSTO1-1 can be used to ameliorate the inflammatory response to LPS. Our findings demonstrate the potential therapeutic utility of GSTO1-1 inhibitors in the modulation of inflammation and suggest their possible application in the treatment of a range of inflammatory conditions.This work was supported by a grant from the Gretel and Gordon Bootes Medical Research Foundation to D.M. and P.B. The National Health and Medical Research Council of Australia (NHMRC) is thanked for Project Grant APP1124673 to PB, MC, LO, AO, and Fellowship support for J.B. (2012–2016 Senior Research Fellowship #1020411). J.B. acknowledges the Australian Federal Government Education Investment Fund Super Science Initiative and the Victorian State Government, Victoria Science Agenda Investment Fund for infrastructure support, and Translating Health Discovery (THD) NCRIS soft infrastructure support through Terapeutic Innovation Australia (TIA)

Non-canonical Wnt signaling and N-cadherin related beta-catenin signaling play a role in mechanically induced osteogenic cell fate.

Understanding how the mechanical microenvironment influences cell fate, and more importantly, by what molecular mechanisms, will enhance not only the knowledge of mesenchymal stem cell biology but also the field of regenerative medicine. Mechanical stimuli, specifically loading induced oscillatory fluid flow, plays a vital role in promoting healthy bone development, homeostasis and morphology. Recent studies suggest that such loading induced fluid flow has the potential to regulate osteogenic differentiation via the upregulation of multiple osteogenic genes; however, the molecular mechanisms involved in the transduction of a physical signal into altered cell fate have yet to be determined.Using immuno-staining, western blot analysis and luciferase assays, we demonstrate the oscillatory fluid flow regulates beta-catenin nuclear translocation and gene transcription. Additionally, real time RT-PCR analysis suggests that flow induces Wnt5a and Ror2 upregulation, both of which are essential for activating the small GTPase, RhoA, upon flow exposure. Furthermore, although beta-catenin phosphorylation is not altered by flow, its association with N-cadherin is, indicating that flow-induced beta-catenin signaling is initiated by adherens junction signaling.We propose that the mechanical microenvironment of bone has the potential to regulate osteogenic differentiation by initiating multiple key molecular pathways that are essential for such lineage commitment. Specifically, non-canonical Wnt5a signaling involving Ror2 and RhoA as well as N-cadherin mediated beta-catenin signaling are necessary for mechanically induced osteogenic differentiation

Reduced cerebral Th17 cell migration confers protection to EAE in DOCK8 deficient mice

Background: Experimental autoimmune encephalomyelitis (EAE) is a murine model of multiple sclerosis mediated by CD4+IL-17a producing (Th17) cells. Mice lacking functional dedicator of cytokinesis 8 (DOCK8) have elevated Th17 cells but do not develop EAE. How DOCK8, a guanine exchange factor expressed by immune cells confers protection to EAE is unknown. Objective: We assessed the migration and functional capacity of Th17 cells in vivo in DOCK8 mutant mice. We also further examined the role of DOCK8 on Th17 cell function ex vivo in both the steady state and in a neuroinflammatory setting. Design Methods: Dock8-/- mice, lacking functional DOCK8 protein were immunised with a neural peptide to induce EAE. CNS infiltrating lymphocytes were quantitated using flow cytometry. Adoptive cell transfer was used to assess T cell migration. T cells isolated from protected mice were tested for their ability to undergo cell differentiation in vitro using cell culture and flow cytometry. Western blot and qPCR were used to analyse protein and RNA expression by cell subsets of DOCK8 mutant mice. Results: In vitro T helper cell differentiation confirmed that the elevated Th17 cell population was not based on a T cell intrinsic differentiation bias or altered apoptosis. Th17 cells expressed normal Th17 cell specific CCR6 levels and migrated towards chemokine gradients in transwell assays. Adoptive transfers of Th1 and Th17 cells in EAE mice indicated a Th17 cell specific migration defect in the absence of functional DOCK8. Conclusions: Mice lacking functional DOCK8 are protected from EAE by reduced CNS infiltration of CD4 T cells. Despite increased resting Th17 cells and normal in vitro differentiation, DOCK8 deficient mice have dysfunctional Th17 cell migration in EAE. DOCK8 thus appears to function by regulating Th17 cell migration into inflamed CNS tissue

Dichloroacetic acid up-regulates hepatic glutathione synthesis via the induction of glutamate-cysteine ligase

Dichloroacetic acid (DCA) has potential for use in cancer therapy and the treatment of metabolic acidosis. However, DCA can create a deficiency of glutathione transferase Zeta (GSTZ1-1). Gstz1 knockout mice have elevated oxidative stress and low glutathione levels that increases their sensitivity to acetaminophen toxicity. As it is highly likely that patients that are treated with DCA will develop drug induced GSTZ1-1 deficiency we considered they could be at risk of elevated toxicity if they are exposed to other drugs that cause oxidative stress or consume glutathione (GSH). To test this hypothesis we treated mice with DCA and acetaminophen (APAP). Surprisingly, the mice pre-treated with DCA suffered less APAP-mediated hepatotoxicity than untreated mice. This protection is most likely due to an increased capacity for the liver to synthesize GSH, since DCA increased the expression and activity of glutamate-cysteine ligase GCL, the rate-limiting enzyme of GSH synthesis. Other pathways for acetaminophen disposal were unchanged or diminished by DCA. Pre-treatment with DCA may be of use in other settings where the maintenance of protective levels of GSH are required. However, DCA may lower the efficacy of drugs that rely on oxidative stress and the depletion of GSH to enhance their cytotoxicity or of drugs that are detoxified by GSH conjugation. Consequently, as the use of DCA in the clinic is likely to increase, it will be critical to evaluate the interactions of DCA with other drugs to ensure the combinations retain their efficacy and do not cause enhanced toxicity

Wnt5a and β-catenin signaling are both necessary for flow induced Runx2 upregulation.

<p>(A) Flow induced Runx2 expression was upregulated 2.2±0.2-fold in scrambled siRNA treated cells exposed to flow verses scrambled siRNA control cells. This fold increase was significantly different (p<0.05) than Wnt5a siRNA treated cells, in which the flow induced Runx2 expression was abrogated. (B) The fold change in Runx2 expression with flow was significantly different between untreated cells and cells with inhibited β-catenin signaling via endostatin treatment (p<0.01). Untreated cells exposed to oscillatory fluid flow had a 2.8±0.5-fold increase in Runx2 expression over control cells; while endostatin treated cells resulted in no difference between flowed and control cells. (C) Western blot analysis demonstrates that there is a significant decrease in β-catenin levels after a 24 hour incubation with endostatin. (Error bars: SEM (n≥6)).</p