Restoration of TET2 Function Blocks Aberrant Self-Renewal and Leukemia Progression.

Loss-of-function mutations in TET2 occur frequently in patients with clonal hematopoiesis, myelodysplastic syndrome (MDS), and acute myeloid leukemia (AML) and are associated with a DNA hypermethylation phenotype. To determine the role of TET2 deficiency in leukemia stem cell maintenance, we generated a reversible transgenic RNAi mouse to model restoration of endogenous Tet2 expression. Tet2 restoration reverses aberrant hematopoietic stem and progenitor cell (HSPC) self-renewal in vitro and in vivo. Treatment with vitamin C, a co-factor of Fe2+ and α-KG-dependent dioxygenases, mimics TET2 restoration by enhancing 5-hydroxymethylcytosine formation in Tet2-deficient mouse HSPCs and suppresses human leukemic colony formation and leukemia progression of primary human leukemia PDXs. Vitamin C also drives DNA hypomethylation and expression of a TET2-dependent gene signature in human leukemia cell lines. Furthermore, TET-mediated DNA oxidation induced by vitamin C treatment in leukemia cells enhances their sensitivity to PARP inhibition and could provide a safe and effective combination strategy to selectively target TET deficiency in cancer. PAPERCLIP.

HOTTIP-dependent R-loop formation regulates CTCF boundary activity and TAD integrity in leukemia.

HOTTIP lncRNA is highly expressed in acute myeloid leukemia (AML) driven by MLL rearrangements or NPM1 mutations to mediate HOXA topologically associated domain (TAD) formation and drive aberrant transcription. However, the mechanism through which HOTTIP accesses CCCTC-binding factor (CTCF) chromatin boundaries and regulates CTCF-mediated genome topology remains unknown. Here, we show that HOTTIP directly interacts with and regulates a fraction of CTCF-binding sites (CBSs) in the AML genome by recruiting CTCF/cohesin complex and R-loop-associated regulators to form R-loops. HOTTIP-mediated R-loops reinforce the CTCF boundary and facilitate formation of TADs to drive gene transcription. Either deleting CBS or targeting RNase H to eliminate R-loops in the boundary CBS of ß-catenin TAD impaired CTCF boundary activity, inhibited promoter/enhancer interactions, reduced ß-catenin target expression, and mitigated leukemogenesis in xenograft mouse models with aberrant HOTTIP expression. Thus, HOTTIP-mediated R-loop formation directly reinforces CTCF chromatin boundary activity and TAD integrity to drive oncogene transcription and leukemia development.

Loss of BRD4 induces cell senescence in HSC/HPCs by deregulating histone H3 clipping.

Bromodomain-containing protein 4 (BRD4) is overexpressed and functionally implicated in various myeloid malignancies. However, the role of BRD4 in normal hematopoiesis remains largely unknown. Here, utilizing an inducible Brd4 knockout mouse model, we find that deletion of Brd4 (Brd4Δ/Δ ) in the hematopoietic system impairs hematopoietic stem cell (HSC) self-renewal and differentiation, which associates with cell cycle arrest and senescence. ATAC-seq analysis shows increased chromatin accessibility in Brd4Δ/Δ hematopoietic stem/progenitor cells (HSC/HPCs). Genome-wide mapping with cleavage under target and release using nuclease (CUT&RUN) assays demonstrate that increased global enrichment of H3K122ac and H3K4me3 in Brd4Δ/Δ HSC/HPCs is associated with the upregulation of senescence-specific genes. Interestingly, Brd4 deletion increases clipped H3 (cH3) which correlates with the upregulation of senescence-specific genes and results in a higher frequency of senescent HSC/HPCs. Re-expression of BRD4 reduces cH3 levels and rescues the senescence rate in Brd4Δ/Δ HSC/HPCs. This study unveils an important role of BRD4 in HSC/HPC function by preventing H3 clipping and suppressing senescence gene expression.

TATDN2 resolution of R-loops is required for survival of BRCA1-mutant cancer cells.

BRCA1-deficient cells have increased IRE1 RNase, which degrades multiple microRNAs. Reconstituting expression of one of these, miR-4638-5p, resulted in synthetic lethality in BRCA1-deficient cancer cells. We found that miR-4638-5p represses expression of TATDN2, a poorly characterized member of the TATD nuclease family. We discovered that human TATDN2 has RNA 3' exonuclease and endonuclease activity on double-stranded hairpin RNA structures. Given the cleavage of hairpin RNA by TATDN2, and that BRCA1-deficient cells have difficulty resolving R-loops, we tested whether TATDN2 could resolve R-loops. Using in vitro biochemical reconstitution assays, we found TATDN2 bound to R-loops and degraded the RNA strand but not DNA of multiple forms of R-loops in vitro in a Mg2+-dependent manner. Mutations in amino acids E593 and E705 predicted by Alphafold-2 to chelate an essential Mg2+ cation completely abrogated this R-loop resolution activity. Depleting TATDN2 increased cellular R-loops, DNA damage and chromosomal instability. Loss of TATDN2 resulted in poor replication fork progression in the presence of increased R-loops. Significantly, we found that TATDN2 is essential for survival of BRCA1-deficient cancer cells, but much less so for cognate BRCA1-repleted cancer cells. Thus, we propose that TATDN2 is a novel target for therapy of BRCA1-deficient cancers.

Long noncoding RNAs: emerging regulators of normal and malignant hematopoiesis.

Genome-wide analyses have revealed that long noncoding RNAs (lncRNAs) are not only passive transcription products, but also major regulators of genome structure and transcription. In particular, lncRNAs exert profound effects on various biological processes, such as chromatin structure, transcription, RNA stability and translation, and protein degradation and localization, that depend on their localization and interacting partners. Recent studies have revealed that thousands of lncRNAs are aberrantly expressed in various cancer types, and some are associated with malignant transformation. Despite extensive efforts, the diverse functions of lncRNAs and molecular mechanisms in which they act remain elusive. Many hematological disorders and malignancies primarily result from genetic alterations that lead to the dysregulation of gene regulatory networks required for cellular proliferation and differentiation. Consequently, a growing list of lncRNAs has been reported to be involved in the modulation of hematopoietic gene expression networks and hematopoietic stem and progenitor cell (HSPC) function. Dysregulation of some of these lncRNAs has been attributed to the pathogenesis of hematological malignancies. In this review, we summarize current advances and knowledge of lncRNAs in gene regulation, focusing on recent progress on the role of lncRNAs in CTCF/cohesin-mediated 3-dimensional genome organization and how such genome folding signals, in turn, regulate transcription, HSPC function, and transformation. This knowledge will provide mechanistic and translational insights into HSPC biology and myeloid malignancy pathophysiology.

Hepatic CYP2B10 is highly induced by binge ethanol and contributes to acute-on-chronic alcohol-induced liver injury.

BACKGROUND: The chronic-plus-binge model of ethanol consumption, where chronically (8-week) ethanol-fed mice are gavaged a single dose of ethanol (E8G1), is known to induce steatohepatitis in mice. However, how chronically ethanol-fed mice respond to multiple binges of ethanol remains unknown. METHODS: We extended the E8G1 model to three gavages of ethanol (E8G3) spaced 24 h apart, sacrificed each group 9 h after the final gavage, analyzed liver injury, and examined gene expression changes using microarray analyses in each group to identify mechanisms contributing to liver responses to binge ethanol. RESULTS: Surprisingly, E8G3 treatment induced lower levels of liver injury, steatosis, inflammation, and fibrosis as compared to mice after E8G1 treatment. Microarray analyses identified several pathways that may contribute to the reduced liver injury after E8G3 treatment compared to E8G1 treatment. The gene encoding cytochrome P450 2B10 (Cyp2b10) was one of the top upregulated genes in the E8G1 group and was further upregulated in the E8G3 group, but only moderately induced after chronic ethanol consumption, as confirmed by RT-qPCR and western blot analyses. Genetic disruption of Cyp2b10 worsened liver injury in E8G1 and E8G3 mice with higher blood ethanol levels compared to wild-type control mice, while in vitro experiments revealed that CYP2b10 did not directly promote ethanol metabolism. Metabolomic analyses revealed significant differences in hepatic metabolites from E8G1-treated Cyp2b10 knockout and WT mice, and these metabolic alterations may contribute to the reduced liver injury in Cyp2b10 knockout mice. CONCLUSION: Hepatic Cyp2b10 expression is highly induced after ethanol binge, and such upregulation reduces acute-on-chronic ethanol-induced liver injury via the indirect modification of ethanol metabolism.

Chalcone derivatives ameliorate lipopolysaccharide-induced acute lung injury and inflammation by targeting MD2.

Acute lung injury (ALI) and its severe form acute respiratory distress syndrome (ARDS) are known as the common causes of respiratory failure in critically ill patients. Myeloid differentiation 2 (MD2), a co-receptor of toll like receptor 4 (TLR4), plays an important role in LPS-induced ALI in mice. Since MD2 inhibition by pharmacological inhibitors or gene knockout significantly attenuates ALI in animal models, MD2 has become an attractive target for the treatment of ALI. In this study we identified two chalcone-derived compounds, 7w and 7x, as new MD2 inhibitors, and investigated the therapeutic effects of 7x and 7w in LPS-induced ALI mouse model. In molecular docking analysis we found that 7w and 7x, formed pi-pi stacking interactions with Phe151 residue of the MD2 protein. The direct binding was confirmed by surface plasmon resonance analysis (with KD value of 96.2 and 31.2 µM, respectively) and by bis-ANS displacement assay. 7w and 7x (2.5, 10 µM) also dose-dependently inhibited the interaction between lipopolysaccharide (LPS) and rhMD2 and LPS-MD2-TLR4 complex formation. In mouse peritoneal macrophages, 7w and 7x (1.25-10 µM) dose-dependently inhibited LPS-induced inflammatory responses, MAPKs (JNK, ERK and P38) phosphorylation as well as NF-κB activation. Finally, oral administration of 7w or 7x (10 mg ·kg-1 per day, for 7 days prior LPS challenge) in ALI mouse model significantly alleviated LPS-induced lung injury, pulmonary edema, lung permeability, inflammatory cells infiltration, inflammatory cytokines expression and MD2/TLR4 complex formation. In summary, we identify 7w and 7x as new MD2 inhibitors to inhibit inflammatory response both in vitro and in vivo, proving the therapeutic potential of 7w and 7x for ALI and inflammatory diseases.

Interleukin-20 exacerbates acute hepatitis and bacterial infection by downregulating IκBζ target genes in hepatocytes.

BACKGROUND & AIMS: Interleukin (IL)-20 and IL-22 belong to the IL-10 family. IL-10 is a well-documented anti-inflammatory cytokine while IL-22 is well known for epithelial protection and its antibacterial function, showing great therapeutic potential for organ damage; however, the function of IL-20 remains largely unknown. METHODS: Il20 knockout (Il20-/-) mice and wild-type littermates were generated and injected with Concanavalin A (ConA) and Klebsiella pneumoniae (K.P.) to induce acute hepatitis and bacterial infection, respectively. RESULTS: Il20-/- mice were resistant to acute hepatitis and exhibited selectively elevated levels of the hepatoprotective cytokine IL-6. Such selective inhibition of IL-6 by IL-20 was due to IL-20 targeting hepatocytes that produce high levels of IL-6 but a limited number of other cytokines. Mechanistically, IL-20 upregulated NAD(P)H: quinone oxidoreductase 1 (NQO1) expression and subsequently promoted the protein degradation of transcription factor IκBζ, resulting in selective downregulation of the IκBζ-dependent gene Il6 as well several other IκBζ-dependent genes including lipocalin-2 (Lcn2). Given the important role of IL-6 and LCN2 in limiting bacterial infection, we examined the effect of IL-20 on bacterial infection and found Il20-/- mice were resistant to K.P. infection and exhibited elevated levels of hepatic IκBζ-dependent antibacterial genes. Moreover, IL-20 upregulated hepatic NQO1 by binding to IL-22R1/IL-20R2 and activating ERK/p38MAPK/NRF2 signaling pathways. Finally, the levels of hepatic IL1B, IL20, and IκBζ target genes were elevated, and correlated with each other, in patients with severe alcoholic hepatitis. CONCLUSIONS: IL-20 selectively inhibits hepatic IL-6 production rather than exerting IL-10-like broad anti-inflammatory properties. Unlike IL-22, IL-20 aggravates acute hepatitis and bacterial infection. Thus, anti-IL-20 therapy could be a promising option to control acute hepatitis and bacterial infection. LAY SUMMARY: Several interleukin (IL)-20 family cytokines have been shown to play important roles in controllimg inflammatory responses, infection and tissue damage, but the role of IL-20 remains unclear. Herein, we elucidated the role of IL-20 in liver disease and bacterial infection. We show that IL-20 can aggravate hepatitis and bacterial infection; thus, targeting IL-20 holds promise for the treatment of patients with liver disease.

CDK9 inhibition improves diabetic nephropathy by reducing inflammation in the kidneys.

Diabetic nephropathy (DN) is a chronic inflammatory renal disease induced by hyperglycemia. Recent studies have implicated cyclin-dependent kinase 9 (CDK9) in inflammatory responses and renal fibrosis. In this study, we explored a potential role of CDK9 in DN by using cultured mouse mesangial cell line SV40 MES-13 and streptozotocin-induced type 1 mouse model of diabetes. We inhibited CDK9 in mice and in cultured cells by a highly selective CDK9 inhibitor, LDC000067 (LDC), and evaluated inflammatory and fibrogenic outcome by mRNA and protein analyses. Our studies show that treatment of diabetic mice with LDC significantly inhibits the levels of inflammatory cytokines and fibrogenic genes in kidney specimens. These reductions were associated with improved renal function. We also found that LDC treatment suppressed MAPK-AP1 activation. We then confirmed the involvement of CDK9 in cultured SV40 MES-13 cells and showed that deficiency in CDK9 prevents glucose-induced inflammatory and fibrogenic proteins. This protection was also afforded by suppression of MAPK-AP1. Taken together, our results how that hyperglycemia activates CDK9-MAPK-AP1 axis in kidneys to induce inflammation and fibrosis, leading to renal dysfunction. Our findings also suggest that CDK9 may serve as a potential therapeutic target for DN.

Characterization of silver carp myosin glycated with phosphorylated konjac oligo-glucomannan.

BACKGROUND: Myosin (Ms) is abundant in fish meat, but it has limited application in the food industry because of its low solubility and thermal stability. Our previous reports found that these functional properties of Ms can be significantly improved after glycation with konjac oligo-glucomannan (KOG). However, the effects of phosphorylated KOG (PKOG) on physicochemical, structural and functional properties of silver carp Ms are still unknown. RESULTS: This study characterized the silver carp Ms protein glycated with PKOG at 50 °C and 75% relative humidity for 48 h. As degree of phosphorylation increased, free amino content increased, whereas degree of grafting decreased. Meanwhile, isoelectric point (pI) reduced, however, PKOGs showed no differences in pI. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis suggested the formation of glycoconjugates, and scanning electron microscopy revealed thinner flakes and uneven appearance of glycoconjugates. Fourier transform infrared spectroscopy indicated that the amide I, II and III bands of Ms were changed by the glycation. Ms became highly soluble in 0.5 mol L-1 NaCl with increased phosphate addition in PKOGs. Thermal stability of Ms was effectively improved when heated at 80 °C for 60 min. CONCLUSION: Glycation with appropriate PKOG might be a promising method for Ms modification because of the resulting improvement in solubility and thermal stability. © 2021 Society of Chemical Industry.

A transcriptional landscape of 28 porcine tissues obtained by super deepSAGE sequencing.

BACKGROUND: Gene expression regulators identified in transcriptome profiling experiments may serve as ideal targets for genetic manipulations in farm animals. RESULTS: In this study, we developed a gene expression profile of 76,000+ unique transcripts for 224 porcine samples from 28 tissues collected from 32 animals using Super deepSAGE technology. Excellent sequencing depth was achieved for each multiplexed library, and replicated samples from the same tissues clustered together, demonstrating the high quality of Super deepSAGE data. Comparison with previous research indicated that our results not only have good reproducibility but also have greatly extended the coverage of the sample types as well as the number of genes. Clustering analysis revealed ten groups of genes showing distinct expression patterns among these samples. Our analysis of over-represented binding motifs identified 41 regulators, and we demonstrated a potential application of this dataset in infectious diseases and immune biology research by identifying an LPS-dependent transcription factor, runt-related transcription factor 1 (RUNX1), in peripheral blood mononuclear cells (PBMCs). The selected genes are specifically responsible for the transcription of toll-like receptor 2 (TLR2), lymphocyte-specific protein tyrosine kinase (LCK), and vav1 oncogene (VAV1), which belong to the T and B cell signaling pathways. CONCLUSIONS: The Super deepSAGE technology and tissue-differential expression profiles are valuable resources for investigating the porcine gene expression regulation. The identified RUNX1 target genes belong to the T and B cell signaling pathways, making them novel potential targets for the diagnosis and therapy of bacterial infections and other immune disorders.

MicroRNA-223 Ameliorates Nonalcoholic Steatohepatitis and Cancer by Targeting Multiple Inflammatory and Oncogenic Genes in Hepatocytes.

Nonalcoholic fatty liver disease (NAFLD) represents a spectrum of diseases ranging from simple steatosis to more severe forms of liver injury including nonalcoholic steatohepatitis (NASH), fibrosis, and hepatocellular carcinoma (HCC). In humans, only 20%-40% of patients with fatty liver progress to NASH, and mice fed a high-fat diet (HFD) develop fatty liver but are resistant to NASH development. To understand how simple steatosis progresses to NASH, we examined hepatic expression of anti-inflammatory microRNA-223 (miR-223) and found that this miRNA was highly elevated in hepatocytes in HFD-fed mice and in human NASH samples. Genetic deletion of miR-223 induced a full spectrum of NAFLD in long-term HFD-fed mice including steatosis, inflammation, fibrosis, and HCC. Furthermore, microarray analyses revealed that, compared to wild-type mice, HFD-fed miR-223 knockout (miR-223KO) mice had greater hepatic expression of many inflammatory genes and cancer-related genes, including (C-X-C motif) chemokine 10 (Cxcl10) and transcriptional coactivator with PDZ-binding motif (Taz), two well-known factors that promote NASH development. In vitro experiments demonstrated that Cxcl10 and Taz are two downstream targets of miR-223 and that overexpression of miR-223 reduced their expression in cultured hepatocytes. Hepatic levels of miR-223, CXCL10, and TAZ mRNA were elevated in human NASH samples, which positively correlated with hepatic levels of several miR-223 targeted genes as well as several proinflammatory, cancer-related, and fibrogenic genes. Conclusion: HFD-fed miR-223KO mice develop a full spectrum of NAFLD, representing a clinically relevant mouse NAFLD model; miR-223 plays a key role in controlling steatosis-to-NASH progression by inhibiting hepatic Cxcl10 and Taz expression and may be a therapeutic target for the treatment of NASH.

Gain of function of ASXL1 truncating protein in the pathogenesis of myeloid malignancies.

Additional Sex Combs-Like 1 (ASXL1) is mutated at a high frequency in all forms of myeloid malignancies associated with poor prognosis. We generated a Vav1 promoter-driven Flag-Asxl1Y588X transgenic mouse model, Asxl1Y588X Tg, to express a truncated FLAG-ASXL1aa1-587 protein in the hematopoietic system. The Asxl1Y588X Tg mice had an enlarged hematopoietic stem cell (HSC) pool, shortened survival, and predisposition to a spectrum of myeloid malignancies, thereby recapitulating the characteristics of myeloid malignancy patients with ASXL1 mutations. ATAC- and RNA-sequencing analyses revealed that the ASXL1aa1-587 truncating protein expression results in more open chromatin in cKit+ cells compared with wild-type cells, accompanied by dysregulated expression of genes critical for HSC self-renewal and differentiation. Liquid chromatography-tandem mass spectrometry and coimmunoprecipitation experiments showed that ASXL1aa1-587 acquired an interaction with BRD4. An epigenetic drug screening demonstrated a hypersensitivity of Asxl1Y588X Tg bone marrow cells to BET bromodomain inhibitors. This study demonstrates that ASXL1aa1-587 plays a gain-of-function role in promoting myeloid malignancies. Our model provides a powerful platform to test therapeutic approaches of targeting the ASXL1 truncation mutations in myeloid malignancies.

CTCF boundary remodels chromatin domain and drives aberrant HOX gene transcription in acute myeloid leukemia.

HOX gene dysregulation is a common feature of acute myeloid leukemia (AML). The molecular mechanisms underlying aberrant HOX gene expression and associated AML pathogenesis remain unclear. The nuclear protein CCCTC-binding factor (CTCF), when bound to insulator sequences, constrains temporal HOX gene-expression patterns within confined chromatin domains for normal development. Here, we used targeted pooled CRISPR-Cas9-knockout library screening to interrogate the function of CTCF boundaries in the HOX gene loci. We discovered that the CTCF binding site located between HOXA7 and HOXA9 genes (CBS7/9) is critical for establishing and maintaining aberrant HOXA9-HOXA13 gene expression in AML. Disruption of the CBS7/9 boundary resulted in spreading of repressive H3K27me3 into the posterior active HOXA chromatin domain that subsequently impaired enhancer/promoter chromatin accessibility and disrupted ectopic long-range interactions among the posterior HOXA genes. Consistent with the role of the CBS7/9 boundary in HOXA locus chromatin organization, attenuation of the CBS7/9 boundary function reduced posterior HOXA gene expression and altered myeloid-specific transcriptome profiles important for pathogenesis of myeloid malignancies. Furthermore, heterozygous deletion of the CBS7/9 chromatin boundary in the HOXA locus reduced human leukemic blast burden and enhanced survival of transplanted AML cell xenograft and patient-derived xenograft mouse models. Thus, the CTCF boundary constrains the normal gene-expression program, as well as plays a role in maintaining the oncogenic transcription program for leukemic transformation. The CTCF boundaries may serve as novel therapeutic targets for the treatment of myeloid malignancies.

A novel salviadione derivative, compound 15a, attenuates diabetes-induced renal injury by inhibiting NF-κB-mediated inflammatory responses.

Diabetic nephropathy is the leading cause of renal failure worldwide. Elevated inflammatory signaling has been shown to lead to deterioration of renal function in human and experimental diabetes. We recently developed a salviadione derivative (compound 15a) that prevented microbial lipopolysaccharide-induced inflammatory responses, which are largely driven by nuclear factor-κB (NF-κB). In the present study, we have tested the hypothesis that 15a will protect kidneys from diabetes-induced dysfunction by suppressing NF-κB activation and inflammatory signaling. Treatment of diabetic mice with 15a inhibited diabetes-induced renal fibrosis, NF-κB activation, and upregulation of proinflammatory cytokines. Histologically, kidney specimens from diabetic mice treated with 15a were indistinguishable from non-diabetic controls. We confirmed our findings in cultured renal tubular epithelial cells exposed to high levels of glucose. In these cultured cells, 15a pretreatment prevented high glucose-induced NF-κB activation and expression of inflammatory cytokines. These protective effects were also reflected in reduced levels of proteins involved in matrix expansion. Overall, our studies show that a salviadione derivative, 15a, is effective in suppressing diabetes-induced NF-κB activation and inflammatory signaling.

Alcohol inhibits T-cell glucose metabolism and hepatitis in ALDH2-deficient mice and humans: roles of acetaldehyde and glucocorticoids.

OBJECTIVE: Aldehyde dehydrogenase 2 (ALDH2), a key enzyme to detoxify acetaldehyde in the liver, exists in both active and inactive forms in humans. Individuals with inactive ALDH2 accumulate acetaldehyde after alcohol consumption. However, how acetaldehyde affects T-cell hepatitis remains unknown. DESIGN: Wild-type (WT) and Aldh2 knockout (Aldh2-/-) mice were subjected to chronic ethanol feeding and concanavalin A (ConA)-induced T-cell hepatitis. Effects of acetaldehyde on T-cell glucose metabolism were investigated in vitro. Human subjects were recruited for binge drinking and plasma cortisol and corticosterone measurement. RESULTS: Ethanol feeding exacerbated ConA-induced hepatitis in WT mice but surprisingly attenuated it in Aldh2-/- mice despite higher acetaldehyde levels in Aldh2-/- mice. Elevation of serum cytokines and their downstream signals in the liver post-ConA injection was attenuated in ethanol-fed Aldh2-/- mice compared to WT mice. In vitro exposure to acetaldehyde inhibited ConA-induced production of several cytokines without affecting their mRNAs in mouse splenocytes. Acetaldehyde also attenuated interferon-γ production in phytohaemagglutinin-stimulated human peripheral lymphocytes. Mechanistically, acetaldehyde interfered with glucose metabolism in T cells by inhibiting aerobic glycolysis-related signal pathways. Finally, compared to WT mice, ethanol-fed Aldh2-/- mice had higher levels of serum corticosterone, a well-known factor that inhibits aerobic glycolysis. Blockade of corticosterone partially restored ConA-mediated hepatitis in ethanol-fed Aldh2-/- mice. Acute alcohol drinking elevated plasma cortisol and corticosterone levels in human subjects with higher levels in those with inactive ALDH2 than those with active ALDH2. CONCLUSIONS: ALDH2 deficiency is associated with elevated acetaldehyde and glucocorticoids post-alcohol consumption, thereby inhibiting T-cell activation and hepatitis.

Wnt1 inhibits vascular smooth muscle cell calcification by promoting ANKH expression.

AIMS: Wnt signaling plays a critical role in vascular calcification (VC). Wnt factors induce different physiological and pathological effects on cardiovascular functions. Wnt1, a ligand of Wnt/ß-catenin signaling, promotes pro-angiogenesis and reduces myocardial infarction. The role of Wnt1 on VC in chronic kidney disease (CKD) is not fully understood. METHODS AND RESULTS: We used human vascular smooth muscle cells (VSMCs) and a rat model of chronic renal failure (CRF), and observed a native protective mechanism by which VC is reduced via the activation of Wnt1 and its transcriptional target ANKH inorganic pyrophosphate transport regulator (ANKH) gene. ANKH is an essential calcification inhibitor that effluxes inorganic pyrophosphate (PPi) from VSMCs to play an inhibitory role in VC. Vascular ANKH and plasma PPi were significantly downregulated in the rat model of CRF. The knockdown or inhibition of ANKH reversed the effect of Wnt1 on VC in VSMCs. Clinical analysis revealed low plasma levels of Wnt1 and PPi were associated with CKD in patients. Applying a Wnt/ß-catenin signaling agonist can alleviate the progression of VC. CONCLUSION: This work reveals the ANKH regulation of Wnt1 in VSMCs is essential for blocking VC. Our findings may contribute to the development of medications that target Wnt signaling and/or ANKH to inhibit VC.

Cardiac Sca-1+ Cells Are Not Intrinsic Stem Cells for Myocardial Development, Renewal, and Repair.

BACKGROUND: For more than a decade, Sca-1+ cells within the mouse heart have been widely recognized as a stem cell population with multipotency that can give rise to cardiomyocytes, endothelial cells, and smooth muscle cells in vitro and after cardiac grafting. However, the developmental origin and authentic nature of these cells remain elusive. METHODS: Here, we used a series of high-fidelity genetic mouse models to characterize the identity and regenerative potential of cardiac resident Sca-1+ cells. RESULTS: With these novel genetic tools, we found that Sca-1 does not label cardiac precursor cells during early embryonic heart formation. Postnatal cardiac resident Sca-1+ cells are in fact a pure endothelial cell population. They retain endothelial properties and exhibit minimal cardiomyogenic potential during development, normal aging and upon ischemic injury. CONCLUSIONS: Our study provides definitive insights into the nature of cardiac resident Sca-1+ cells. The observations challenge the current dogma that cardiac resident Sca-1+ cells are intrinsic stem cells for myocardial development, renewal, and repair, and suggest that the mechanisms of transplanted Sca-1+ cells in heart repair need to be reassessed.

Six1 regulates leukemia stem cell maintenance in acute myeloid leukemia.

Molecular genetic changes in acute myeloid leukemia (AML) play crucial roles in leukemogenesis, including recurrent chromosome translocations, epigenetic/spliceosome mutations and transcription factor aberrations. Six1, a transcription factor of the Sine oculis homeobox (Six) family, has been shown to transform normal hematopoietic progenitors into leukemia in cooperation with Eya. However, the specific role and the underlying mechanism of Six1 in leukemia maintenance remain unexplored. Here, we showed increased expression of SIX1 in AML patients and murine leukemia stem cells (c-Kit+ cells, LSCs). Importantly, we also observed that a higher level of Six1 in human patients predicts a worse prognosis. Notably, knockdown of Six1 significantly prolonged the survival of MLL-AF9-induced AML mice with reduced peripheral infiltration and tumor burden. AML cells from Six1-knockdown (KD) mice displayed a significantly decreased number and function of LSC, as assessed by the immunophenotype, colony-forming ability and limiting dilution assay. Further analysis revealed the augmented apoptosis of LSC and decreased expression of glycolytic genes in Six1 KD mice. Overall, our data showed that Six1 is essential for the progression of MLL-AF9-induced AML via maintaining the pool of LSC.

Hepatocytes and neutrophils cooperatively suppress bacterial infection by differentially regulating lipocalin-2 and neutrophil extracellular traps.

Lipocalin-2 (LCN2), also known as neutrophil gelatinase-associated lipocalin (NGAL), a key antibacterial protein, is highly elevated in patients with end-stage liver disease that is often associated with bacterial infection. LCN2 is expressed at high levels in both hepatocytes and neutrophils; however, how hepatocyte-derived and neutrophil-derived LCN2 cooperate to combat bacterial infection remains unclear. Here, by studying hepatocyte-specific and myeloid-specific Lcn2 knockout mice in two models of systemic and local Klebsiella pneumoniae infections, we demonstrated that hepatocytes played a critical role in controlling systemic infection by secreting LCN2 protein into the circulation following intraperitoneal injection of bacteria, whereas neutrophils were more important in combating local lung infection by carrying LCN2 in their specific granules to the local infection site following intratracheal intubation of bacteria. Both hepatocyte-derived and myeloid cell-derived LCN2 were required against bacterial infection in the peritoneal cavity and liver necrotic areas following intraperitoneal injection of Klebsiella pneumoniae. LCN2/NGAL protein was detected in neutrophil extracellular traps (NETs) in activated neutrophils from mice and humans. Disruption of the Lcn2 gene in neutrophils abolished LCN2 on NETs, whereas deletion of this gene in hepatocytes did not affect LCN2 protein on NETs. Genetic deletion of the Lcn2 gene globally or specifically in neutrophils did not affect NET formation but reduced the bactericidal effect of NETs in vitro. Finally, NGAL-positive NETs were detected in the liver from patients with various types of liver diseases. CONCLUSION: Both hepatocytes and neutrophils combat bacterial infection through the production of LCN2; extracellular LCN2 secreted by hepatocytes limits systemic bacterial infection, whereas neutrophils carry LCN2 protein to the local site and against local bacterial infection through NETs. (Hepatology 2018).