Glutathione (GSH) is the main antioxidant against cell damage. Several pathological states course with reduced nucleophilic tone and perturbation of redox homeostasis due to changes in the 2GSH/GSSG ...ratio. Here, we investigated the regulation of the rate-limiting GSH biosynthetic heterodimeric enzyme γ-glutamyl-cysteine ligase (GCL) by microRNAs (miRNAs).
"In silico" analysis of the 3'- untranslated regions (UTRs) of both catalytic (GCLc) and regulatory (GCLm) subunits of GCL enabled an identification of miR-433 as a strong candidate for the targeting of GCL. Transitory overexpression of miR-433 in human umbilical vein endothelial cells (HUVEC) showed a downregulation of both GCLc and GCLm in a nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-independent manner. Increases in pro-oxidant stimuli such as exposure to hydrogen peroxide or GSH depletion in endothelial and hepatic cells caused an expected increase in GCLc and GCLm protein expression and abrogation of miR-433 levels, thus supporting a cross-regulation of these pathways. Treatment of HUVEC with miR-433 resulted in reduced antioxidant and redox potentials, increased S-glutathionylation, and reduced endothelial nitric oxide synthase activation. In vivo models of renal and hepatic fibrosis were associated with transforming growth factor β1 (TGF-β1)-related reduction of GCLc and GCLm levels that were miR-433 dependent.
We describe for the first time an miRNA, miR-433, capable of directly targeting GCL and promoting functional consequences in endothelial physiology and fibrotic processes by decreasing GSH levels.
Hepatic fibrosis is a global health problem currently without effective therapeutic approaches. Even though the ubiquitin‐like posttranslational modification of neddylation, that conjugates Nedd8 ...(neural precursor cell expressed developmentally downregulated) to specific targets, is aberrant in many pathologies, its relevance in liver fibrosis (LF) remained unexplored. Our results show deregulated neddylation in clinical fibrosis and both in mouse bileductligation– and CCl4‐induced fibrosis. Importantly, neddylation inhibition, by using the pharmacological inhibitor, MLN4924, reduced liver injury, apoptosis, inflammation, and fibrosis by targeting different hepatic cell types. On one hand, increased neddylation was associated with augmented caspase 3 activity in bile‐acid–induced apoptosis in mouse hepatocytes whereas neddylation inhibition ameliorated apoptosis through reduction of expression of the Cxcl1 and Ccl2 chemokines. On the other hand, chemokine receptors and cytokines, usually induced in activated macrophages, were reduced after neddylation inhibition in mouse Kupffer cells. Under these circumstances, decreased hepatocyte cell death and inflammation after neddylation inhibition could partly account for reduction of hepatic stellate cell (HSC) activation. We provide evidence that augmented neddylation characterizes activated HSCs, suggesting that neddylation inhibition could be important for resolving LF by directly targeting these fibrogenic cells. Indeed, neddylation inhibition in activated HSCs induces apoptosis in a process partly mediated by accumulation of c‐Jun, whose cullin‐mediated degradation is impaired under these circumstances. Conclusion: Neddylation inhibition reduces fibrosis, suggesting neddylation as a potential and attractive therapeutic target in liver fibrosis. (Hepatology 2017;65:694‐709).
Inflammation is the hallmark of chronic liver disease. Metabolism is a key determinant to regulate the activation of immune cells. Here, we define the role of sirtuin 1 (SIRT1), a main metabolic ...regulator, in controlling the activation of macrophages during cholestatic liver disease and in response to endotoxin.
We have used mice overexpressing SIRT1, which we treated with intraperitoneal lipopolysaccharides or induced cholestasis by bile duct ligation. Bone marrow–derived macrophages were used for mechanistic in vitro studies. Finally, PEPC-Boy mice were used for adoptive transfer experiments to elucidate the impact of SIRT1-overexpressing macrophages in contributing to cholestatic liver disease.
We found that SIRT1 overexpression promotes increased liver inflammation and liver injury after lipopolysaccharide/GalN and bile duct ligation; this was associated with an increased activation of the inflammasome in macrophages. Mechanistically, SIRT1 overexpression associated with the activation of the mammalian target of rapamycin (mTOR) pathway that led to increased activation of macrophages, which showed metabolic rewiring with increased glycolysis and broken tricarboxylic acid cycle in response to endotoxin in vitro. Activation of the SIRT1/mTOR axis in macrophages associated with the activation of the inflammasome and the attenuation of autophagy. Ultimately, in an in vivo model of cholestatic disease, the transplantation of SIRT1-overexpressing myeloid cells contributed to liver injury and fibrosis.
Our study provides novel mechanistic insights into the regulation of macrophages during cholestatic disease and the response to endotoxin, in which the SIRT1/mTOR crosstalk regulates macrophage activation controlling the inflammasome, autophagy and metabolic rewiring.
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Hematopoietic stem cells (HSC) undergo rapid expansion in response to stress stimuli. Specifically, there is rapid expansion of leukocytes in response to pathogenic bacteria which underpins the ...mammalian response to infection. Presently, the mechanisms by which HSC metabolism is regulated in response to the challenges of pathogenic stress are not fully understood. Here we investigate the bioenergetic processes which facilitate the HSCs expansion in response to infection.
We created 2 animal models to study mitochondrial transfer from bone marrow cells to HSCs. The first is a xenograft humanized mouse model in which human CD34+ cord blood (CB) were transplanted into NSG (huNSG) to study mouse mtDNA transfer into cell sorted humanhematopoietic stem cells (huHSC) (mCD45-, hCD34+, hCD38-, hCD45RA-, hCD90+, hCD49f+). The second took advantage of strain specific SNP in the COX3 and ND3 region between NSG (A/C) and C57BL/6 (G/T) mice. Here we transplanted C57BL/6 derived lineage negative hematopoietic progenitor cells into NSG recipient animals and then the chimeric NSG mice were treated with lipopolysaccharide (LPS). We then isolated the HSCs by cell sorting (LN-, CD117+, Sca1+, CD48-, CD150+ and CD34+) and performed mitochondria analysis on the hematopoietic progenitors. In both transplant models we detected recipient mtDNA in donor HSC post infection (11.2% +/-4.1% of total mtDNA from recipient after LPS treatment).HSC expansion and differentiation (as measured by colony forming cell assay and Ki-67 staining) was shown for Salmonella Typhimurium (S.typhimurium)and LPS treated animals. Seahorse mitochondrial stress analysis confirmed increased oxygen consumption levels in hematopoietic stem and progenitor cells (HSPC) from LPS (2 hours) and S.typhimurium(72 hours) treated C57BL/6. We confirmed that this was not due to mitochondrial biogenesis by showing that TFAM was not upregulated in HSPC in response to LPS at 2 hours.
In previous work we have reported that transfer of functional mitochondria from the BM to AML is driven by AML derived NOX2 dependent ROS (Marlein, Blood 2017). To determine if ROS drives mitochondrial transfer from bone marrow cells to HSCs, we treated huNSG with with L-buthionine-sulfoximine (BSO), a GSH biosynthesis inhibitor, for 2 hours. Mouse mtDNA (12.3% +/-4.8) was detected in huHSC from BSO treated huNSG mice but not in the control huHSC. Moreover, the ROS scavenger N-acetyl-cysteine (NAC) was shown to reduce mtDNA transfer in LPS treated mice. We next investigated which cell of the BM donates their mitochondria during stress hematopoiesis. To do this we cultured bone marrow stromal cells (BMSC), osteoblast and macrophages with HSPC in the presence of H2O2. Transfer of mtDNA was observed from BMSC to HSPC, but we did not see mitochondria transfer from either macrophages or osteoblasts to HSPC. In addition, in vivo mitochondrial levels were reduced in the BMSCs (CD105+, CD140a+ CD31-, Ter119-, CD45-) of LPS treated C57BL/6 compared to control animals and this reduction was inhibited by the addition of NAC. Finally, ROS has been shown to drive PtdIns(3,4,5)P3 (PIP3) oxidation of PTEN and subsequent activation of PI3K (Covey, Oncogene 2007). We foundthat AKT phosphorylation was elevated in both the BMSC and the HSC of LPS treated animals when compared to the untreated controls, andin vivoadministration ofidelalisib (a PI3 Kinase delta inhibiting drug), decreased mitochondrial transfer from the BMSC to HSC.
Our data indicates that infection induced ROS drives PI3 kinase mediated mitochondrial transfer from the BM microenvironment to HSCs. This process is an early physiologic event in the mammalian response to acute bacterial infection and results in bioenergetic changes which underpin emergency granulopoiesis.
Bowles:Janssen: Research Funding; Abbvie: Research Funding. Rushworth:Abbvie: Research Funding; Janssen: Research Funding.
The delivery of pathogens to lysosomes for degradation provides an important defense against infection. Degradation is enhanced when LC3 is conjugated to endosomes and phagosomes containing pathogens ...to facilitate fusion with lysosomes. In phagocytic cells, TLR signaling and Rubicon activate LC3-associated phagocytosis (LAP) where stabilization of the NADPH oxidase leads to sustained ROS production and raised vacuolar pH. Raised pH triggers the assembly of the vacuolar ATPase on the vacuole membrane where it binds ATG16L1 to recruit the core LC3 conjugation complex (ATG16L1:ATG5-12). This V-ATPase-ATG16L1 axis is also activated in nonphagocytic cells to conjugate LC3 to endosomes containing extracellular microbes. Pathogens provide additional signals for recruitment of LC3 when they raise vacuolar pH with pore-forming toxins and proteins, phospholipases, or specialized secretion systems. Many microbes secrete virulence factors to inhibit ROS production and/or the V-ATPase-ATG16L1 axis to slow LC3 recruitment and avoid degradation in lysosomes.
Hu antigen R (HuR) is a central RNA‐binding protein regulating cell dedifferentiation, proliferation, and survival, which are well‐established hallmarks of cancer. HuR is frequently overexpressed in ...tumors correlating with tumor malignancy, which is in line with a role for HuR in tumorigenesis. However, the precise mechanism leading to changes in HuR expression remains unclear. In the liver, HuR plays a crucial role in hepatocyte proliferation, differentiation, and transformation. Here, we unraveled a novel mean of regulation of HuR expression in hepatocellular carcinoma (HCC) and colon cancer. HuR levels correlate with the abundance of the oncogene, murine double minute 2 (Mdm2), in human HCC and colon cancer metastases. HuR is stabilized by Mdm2‐mediated NEDDylation in at least three lysine residues, ensuring its nuclear localization and protection from degradation. Conclusion: This novel Mdm2/NEDD8/HuR regulatory framework is essential for the malignant transformation of tumor cells, which, in turn, unveils a novel signaling paradigm that is pharmacologically amenable for cancer therapy. (Hepatology 2012)
Background & Aims: NEMO is the regulatory subunit of the IκB kinase (IKK) complex and is involved in controlling nuclear factor κB (NF-κB) activation. NEMO knockout mice die during embryogenesis due ...to massive hepatocyte apoptosis. Here we investigated the role of NEMO-dependent signaling in hepatocytes during acute liver injury. Methods: We generated conditional hepatocyte-specific NEMO knockout mice using the loxP system with the Cre recombinase under the control of the albumin promoter (NEMOΔLPC). In these mice, we studied mechanisms of tumor necrosis factor (TNF)- and ischemia/reperfusion-dependent liver cell damage. Results: In adult NEMOΔLPC animals, NEMO is specifically deleted in hepatocytes and no differences in survival, growth, and fertility were found when compared with wild-type (NEMOf/f ) mice. TNF stimulation of NEMOΔLPC mice resulted in high serum transaminase levels and massive hepatocyte apoptosis, which were associated with lack of IκBα degradation, inhibition of NF-κB activation, and target gene transcription. Additionally, ischemia/reperfusion resulted in higher nonparenchymal cell–dependent induction of oxidative stress and stronger inflammation in NEMOΔLPC mice. This led to massive hepatocyte apoptosis and death of the animals, while NEMOf/f mice survived with significantly lesser liver damage, showing mainly necrotic cell death. Thus, complete inhibition of NF-κB activation in hepatocytes, in contrast to attenuation in hepatocyte-specific IKK2−/− mice, determines the type of liver cell damage during ischemia/reperfusion injury and is associated with a poor prognosis. Conclusions: Our results show that understanding of the fine tuning of NF-κB modulation during liver injury is essential to develop new therapeutic strategies.
Glycine N-methyltransferase (GNMT) is the most abundant methyltransferase in the liver and a master regulator of the transmethylation flux. GNMT downregulation leads to loss of liver function ...progressing to fibrosis, cirrhosis, and hepatocellular carcinoma. Moreover, GNMT deficiency aggravates cholestasis-induced fibrogenesis. To date, little is known about the mechanisms underlying downregulation of GNMT levels in hepatic fibrosis and cirrhosis. On this basis, microRNAs are epigenetic regulatory elements that play important roles in liver pathology. In this work, we aim to study the regulation of GNMT by microRNAs during liver fibrosis and cirrhosis. Luciferase assay on the 3'UTR-Gnmt was used to confirm in silico analysis showing that GNMT is potentially targeted by the microRNA miR-873-5p. Correlation between GNMT and miR-873-5p in human cholestasis and cirrhosis together with miR-873-5p inhibition in vivo in different mouse models of liver cholestasis and fibrosis bile duct ligation and Mdr2 (Abcb4)
mouse were then assessed. The analysis of liver tissue from cirrhotic and cholestatic patients, as well as from the animal models, showed that miR-873-5p inversely correlated with the expression of GNMT. Importantly, high circulating miR-873-5p was also detected in cholestastic and cirrhotic patients. Preclinical studies with anti-miR-873-5p treatment in bile duct ligation and Mdr2
mice recovered GNMT levels in association with ameliorated inflammation and fibrosis mainly by counteracting hepatocyte apoptosis and cholangiocyte proliferation. In conclusion, miR-873-5p emerges as a novel marker for liver fibrosis, cholestasis, and cirrhosis and therapeutic approaches based on anti-miR-873-5p may be effective treatments for liver fibrosis and cholestatic liver disease.
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