Gene Ablation Research Articles

Loss of the repressor REST affects progesterone receptor function and promotes uterine leiomyoma pathogenesis

Uterine leiomyomas (UL) are benign tumors that arise in the myometrial layer of the uterus. The standard treatment option for UL is hysterectomy, although hormonal therapies, such as selective progesterone receptor modulators, are often used as temporary treatment options to reduce symptoms or to slow the growth of tumors. However, since the pathogenesis of UL is poorly understood and most hormonal therapies are not based on UL-specific, divergent hormone signaling pathways, hallmarks that predict long-term efficacy and safety of pharmacotherapies remain largely undefined. In a previous study, we reported that aberrant expression of repressor element 1 silencing transcription factor/neuron-restrictive silencing factor (REST/NRSF) target genes activate UL growth due to the near ubiquitous loss of REST. Here, we show that ablation of the Rest gene in mouse uterus leads to UL phenotype and gene-expression patterns analogous to UL, including altered estrogen and progesterone signaling pathways. We demonstrate that many of the genes dysregulated in UL harbor cis-regulatory elements bound by REST and progesterone receptor (PGR) adjacent to each other. Crucially, we identify an interaction between REST and PGR in healthy myometrium and present a putative mechanism for the dysregulation of progesterone-responsive genes in UL ensuing in the loss of REST. Using three Rest conditional knockout mouse lines, we provide a comprehensive picture of the impact loss of REST has in UL pathogenesis and in altering the response of UL to steroid hormones.

Phosphorylation of RXRα mediates the effect of JNK to suppress hepatic FGF21 expression and promote metabolic syndrome

The cJun NH2-terminal kinase (JNK) signaling pathway in the liver promotes systemic changes in metabolism by regulating peroxisome proliferator-activated receptor α (PPARα)-dependent expression of the hepatokine fibroblast growth factor 21 (FGF21). Hepatocyte-specific gene ablation studies demonstrated that the Mapk9 gene (encoding JNK2) plays a key mechanistic role. Mutually exclusive inclusion of exons 7a and 7b yields expression of the isoforms JNK2α and JNK2β. Here we demonstrate that Fgf21 gene expression and metabolic regulation are primarily regulated by the JNK2α isoform. To identify relevant substrates of JNK2α, we performed a quantitative phosphoproteomic study of livers isolated from control mice, mice with JNK deficiency in hepatocytes, and mice that express only JNK2α or JNK2β in hepatocytes. We identified the JNK substrate retinoid X receptor α (RXRα) as a protein that exhibited JNK2α-promoted phosphorylation invivo. RXRα functions as a heterodimeric partner of PPARα and may therefore mediate the effects of JNK2α signaling on Fgf21 expression. To test this hypothesis, we established mice with hepatocyte-specific expression of wild-type or mutated RXRα proteins. We found that the RXRα phosphorylation site Ser260 was required for suppression of Fgf21 gene expression. Collectively, these data establish a JNK-mediated signaling pathway that regulates hepatic Fgf21 expression.

Evaluation of two gene ablation methods for the analysis of pregnenolone function in zebrafish embryonic development

Pregnenolone (P5) is a steroid that functions in the brain and in zebrafish embryogenesis. It is synthesized from cholesterol via the enzymatic activity of P450scc, encoded by CYP11A1. P5 exerts its function by activating CLIP1, which in turn promotes microtubule assembly necessary for many biological processes including embryogenesis. To examine the functional relatedness of CYP11A1 and CLIP1, we ablated the embryonic expression of both genes in zebrafish, i.e. cyp11a1 and clip1a. Two cyp11a1 knockout fish lines were generated. Both homozygous cyp11a1 knockout lines appeared normal. But the development of fish embryos was delayed and embryonic cell migration was reduced when cyp11a1 function was depleted of by morpholinos. This discrepancy in phenotypes by two different gene depletion methods was also observed for clip1a. While clip1a morphants are defective in embryogenesis, clip1a knockout fish appeared normal. The phenotypes depend on the methods that create gene depletion. While knockout fish lines do not have expected phenotypic defects, clip1a and cyp11a1 morpholinos both reduce embryonic cell migration. We have evaluated the usefulness of both methods of gene ablation, and conclude that CYP11A1 and CLIP1 function in the same pathway to promote embryogenesis.

Lack of evidence for participation of TMEM150C in sensory mechanotransduction.

The membrane protein TMEM150C has been proposed to form a mechanosensitive ion channel that is required for normal proprioceptor function. Here, we examined whether expression of TMEM150C in neuroblastoma cells lacking Piezo1 is associated with the appearance of mechanosensitive currents. Using three different modes of mechanical stimuli, indentation, membrane stretch, and substrate deflection, we could not evoke mechanosensitive currents in cells expressing TMEM150C. We next asked if TMEM150C is necessary for the normal mechanosensitivity of cutaneous sensory neurons. We used an available mouse model in which the Tmem150c locus was disrupted through the insertion of a LacZ cassette with a splice acceptor that should lead to transcript truncation. Analysis of these mice indicated that ablation of the Tmem150c gene was not complete in sensory neurons of the dorsal root ganglia (DRG). Using a CRISPR/Cas9 strategy, we made a second mouse model in which a large part of the Tmem150c gene was deleted and established that these Tmem150c-/- mice completely lack TMEM150C protein in the DRGs. We used an ex vivo skin nerve preparation to characterize the mechanosenstivity of mechanoreceptors and nociceptors in the glabrous skin of the Tmem150c-/- mice. We found no quantitative alterations in the physiological properties of any type of cutaneous sensory fiber in Tmem150c-/- mice. Since it has been claimed that TMEM150C is required for normal proprioceptor function, we made a quantitative analysis of locomotion in Tmem150c-/- mice. Here again, we found no indication that there was altered gait in Tmem150c-/- mice compared to wild-type controls. In summary, we conclude that existing mouse models that have been used to investigate TMEM150C function in vivo are problematic. Furthermore, we could find no evidence that TMEM150C forms a mechanosensitive channel or that it is necessary for the normal mechanosensitivity of cutaneous sensory neurons.

Sclerostin ablation prevents aortic valve stenosis in mice.

The objective of this study was to test the hypothesis that targeting sclerostin would accelerate the progression of aortic valve stenosis. Sclerostin (mouse gene, Sost) is a secreted glycoprotein that acts as a potent regulator of bone remodeling. Antibody therapy targeting sclerostin is approved for osteoporosis but results from a stage III clinical trial showed multiple off-target cardiovascular effects. Wild-type (WT, Sost+/+) and Sost-gene knockout-expression (Null, Sost-/-) mice were generated and maintained to 12 mo of age on a high-cholesterol diet to induce aortic valve stenosis. Mice were examined by echocardiography, histology, and RNAseq. Immortalized valve interstitial cells were developed from each genotype for in vitro studies. Null mice developed a bone overgrowth phenotype, similar to patients with sclerosteosis. Surprisingly, however, WT mice developed hemodynamic signs of aortic valve stenosis, whereas Null mice were unchanged. WT mice had thicker aortic valve leaflets and higher amounts of α-smooth muscle actin, a marker myofibroblast activation and dystrophic calcification, with very little evidence of Runx2 expression, a marker of osteogenic calcification. RNAseq analysis of aortic roots indicated the HOX family of transcription factors was significantly upregulated in Null mice, and valve interstitial cells from Null animals were enriched with Hoxa1, Hoxb2, and Hoxd3 subtypes with downregulated Hoxa7. In addition, Null valve interstitial cells were shown to be less contractile than their WT counterparts. Contrary to our hypothesis, sclerostin targeting prevented hallmarks of aortic valve stenosis and indicates that targeted antibody treatments for osteoporosis may be beneficial for these patients regarding aortic stenosis.NEW & NOTEWORTHY We have found that genetic ablation of the Sost gene (protein: sclerostin) prevents aortic valve stenosis in aged, Western diet mice. This is a new role for sclerostin in the cardiovascular system. To the knowledge of the authors, this is one of the first studies directly manipulating sclerostin in a cardiovascular disease model and the first to specifically study the aortic valve. We also provide a potential new role for Hox genes in cardiovascular disease, noting pan-Hox upregulation in the aortic roots of sclerostin genetic knockouts. The role of Hox genes in postnatal cardiovascular health and disease is another burgeoning field of study to which this article contributes.

Combinatorial targeting of G-protein-coupled bile acid receptor 1 and cysteinyl leukotriene receptor 1 reveals a mechanistic role for bile acids and leukotrienes in drug-induced liver injury.

Drug-induced liver injury (DILI) is a common disorder that involves both direct liver cell toxicity and immune activation. The bile acid receptor, G-protein-coupled bile acid receptor 1 (GPBAR1; Takeda G-protein-coupled receptor 5 [TGR5]), and cysteinyl leukotriene receptor (CYSLTR) 1 are G-protein-coupled receptors activated by bile acids and leukotrienes, exerting opposite effects on cell-to-cell adhesion, inflammation, and immune cell activation. To investigate whether GPBAR1 and CYSLTR1 mutually interact in the development of DILI, we developed an orally active small molecule, CHIN117, that functions as a GPBAR1 agonist and CYSLTR1 antagonist. RNA-sequencing analysis of liver explants showed that acetaminophen (APAP) intoxication positively modulates the leukotriene pathway, CYSLTR1, 5-lipoxygenase, and 5-lipoxygenase activating protein, whereas GPBAR1 gene expression was unchanged. In mice, acute liver injury induced by orally dosing APAP (500 mg/kg) was severely exacerbated by Gpbar1 gene ablation and attenuated by anti-Cysltr1 small interfering RNA pretreatment. Therapeutic dosing of wild-type mice with CHIN117 reversed the liver damage caused by APAP and modulated up to 1300 genes, including 38 chemokines and receptors, that were not shared by dosing mice with a selective GPBAR1 agonist or CYSLTR1 antagonist. Coexpression of the two receptors was detected in liver sinusoidal endothelial cells (LSECs), monocytes, and Kupffer cells, whereas combinatorial modulation of CYSLTR1 and GPBAR1 potently reversed LSEC/monocyte interactions. CHIN117 reversed liver damage and liver fibrosis in mice administered CCl 4 . By genetic and pharmacological approaches, we demonstrated that GPBAR1 and CYSLTR1 mutually interact in the development of DILI. A combinatorial approach designed to activate GPBAR1 while inhibiting CYSLTR1 reverses liver injury in models of DILI.

Myeloid cell-specific ablation of Runx2 gene exacerbates post-infarct cardiac remodeling

Runt-related transcription factor 2 (Runx2), a regulator of osteoblast differentiation, is pathologically involved in vascular calcification; however, the significance of Runx2 in cardiac homeostasis remains unclear. Here, we investigated the roles of Runx2 in cardiac remodeling after myocardial infarction (MI). The expression of Runx2 mRNA and protein was upregulated in murine hearts after MI. Runx2 was expressed in heart-infiltrating myeloid cells, especially in macrophages, at the border zone of post-infarct myocardium. To analyze the biological functions of Runx2 in cardiac remodeling, myeloid cell-specific Runx2 deficient (CKO) mice were exposed to MI. After MI, ventricular weight/tibia length ratio was increased in CKO mice, concomitant with severe cardiac dysfunction. Cardiac fibrosis was exacerbated in CKO mice, consistent with the upregulation of collagen 1a1 expression. Mechanistically, immunohistochemical analysis using anti-CD31 antibody showed that capillary density was decreased in CKO mice. Additionally, conditioned culture media of myeloid cells from Runx2 deficient mice exposed to MI induced the tube formation of vascular endothelial cells to a lesser extent than those from control mice. RNA-sequence showed that the expression of pro-angiogenic or anti-angiogenic factors was altered in macrophages from Runx2-deficient mice. Collectively, Runx2+ myeloid cells infiltrate into post-infarct myocardium and prevent adverse cardiac remodeling, at least partially, by regulating endothelial cell function.

259 (PB039) - CDK11 is a selective dependency in neuroblastoma harboring loss of chromosome 1p36

Background: Neuroblastoma is the most common infant cancer and exhibits frequent deletion of chromosome 1p36 in the high-risk setting. We sought to identify key dependencies and drug targets driving high-risk neuroblastoma cell growth. Materials and methods: We used targeted shRNA-mediated gene ablation to identify and validate CDK11 as a drug target in SK-N-BE(2) cells. Used CDK11 nanoBRET assay, radiometric kinase activity assay catalyzed by recombinant kinases, and cDNA complementation using exogenous WT CDK11, inhibitor resistant CDK11G579S and kinase deficient CDK11K467M to determine CDK11 target engagement and activity. Total RNA-seq was deployed to define splicing modulation upon CDK11 perturbation and genome-wide CRISPR-Cas9 screening to define synthetic lethal interactions upon CDK11 perturbation. Results: We discovered that CDK11 (CDC2L1/CDC2L2) is frequently deleted of one copy at chromosome 1p36 and is a deep dependency in high-risk neuroblastoma cells harboring 1p36 Loss-of-heterozygosity (Δ1p36). We show that CDK11 loss-of-function via genetic or chemical inhibition preferentially impedes proliferation of Δ1p36 tumor cells vs WT 1p36 tumor cells. Reconstitution of wild-type CDK11 levels in Δ1p36 tumor cell lines rendered neuroblastoma cells resistant to CDK11 inhibitors whereas kinase-deficient CDK11 did not, suggesting a dosage requirement for CDK11 kinase function in Δ1p36 neuroblastoma cells. Chemical inhibition of CDK11 kinase activity in Δ1p36 tumor cells induces intron-retention splicing defects at mRNA processing and export genes, indicating a core role for CDK11 in RNA metabolism. A genome-wide CRISPR screen for synthetic lethality with CDK11 inhibitors revealed that cognate cyclin CCNL1 and a set of core splicing regulators collaborate with CDK11 to enforce neuroblastoma tumor cell growth. Conclusions: Together, our findings identify CDK11 as a druggable vulnerability and potential therapeutic target for high-risk neuroblastoma patients exhibiting Δ1p36. Conflict of interest: Other Substantive Relationships: All authors are employees of Syros Pharmaceuticals.

Renal Transcriptome and Metabolome in Mice with Principal Cell-Specific Ablation of the Tsc1 Gene: Derangements in Pathways Associated with Cell Metabolism, Growth and Acid Secretion.

Tuberous sclerosis complex (TSC) is caused by mutations in the hamartin (TSC1) or tuberin (TSC2) genes. Using a mouse model of TSC renal cystogenesis that we have previously described, the current studies delineate the metabolic changes in the kidney and their relation to alterations in renal gene expression. To accomplish this, we compared the metabolome and transcriptome of kidneys from 28-day-old wildtype (Wt) and principal cell-specific Tsc1 KO (Tsc1 KO) mice using targeted 1H nuclear magnetic resonance targeted metabolomic and RNA-seq analyses. The significant changes in the kidney metabolome of Tsc1 KO mice included reductions in the level of several amino acids and significant decreases in creatine, NADH, inosine, UDP-galactose, GTP and myo-inositol levels. These derangements may affect energy production and storage, signal transduction and synthetic pathways. The pertinent derangement in the transcriptome of Tsc1 KO mice was associated with increased collecting duct acid secretion, active cell division and the up-regulation of signaling pathways (e.g., MAPK and AKT/PI3K) that suppress the TSC2 GTPase-activating function. The combined renal metabolome and transcriptome alterations observed in these studies correlate with the unregulated growth and predominance of genotypically normal A-intercalated cells in the epithelium of renal cysts in Tsc1 KO mice.

Pathway-specific contribution of parvalbumin interneuron NMDARs to synaptic currents and thalamocortical feedforward inhibition

Prefrontal cortex (PFC) is a site of information convergence important for behaviors relevant to psychiatric disorders. Despite the importance of inhibitory GABAergic parvalbumin-expressing (PV+) interneurons to PFC circuit function and decades of interest in N-methyl-D-aspartate receptors (NMDARs) in these neurons, examples of defined circuit functions that depend on PV+ interneuron NMDARs have been elusive. Indeed, it remains controversial whether all PV+ interneurons contain functional NMDARs in adult PFC, which has major consequences for hypotheses of the pathogenesis of psychiatric disorders. Using a combination of fluorescent in situ hybridization, pathway-specific optogenetics, cell-type-specific gene ablation, and electrophysiological recordings from PV+ interneurons, here we resolve this controversy. We found that nearly 100% of PV+ interneurons in adult medial PFC (mPFC) express transcripts encoding GluN1 and GluN2B, and they have functional NMDARs. By optogenetically stimulating corticocortical and thalamocortical inputs to mPFC, we show that synaptic NMDAR contribution to PV+ interneuron EPSCs is pathway-specific, which likely explains earlier reports of PV+ interneurons without synaptic NMDAR currents. Lastly, we report a major contribution of NMDARs in PV+ interneurons to thalamus-mediated feedforward inhibition in adult mPFC circuits, suggesting molecular and circuit-based mechanisms for cognitive impairment under conditions of reduced NMDAR function. These findings represent an important conceptual advance that has major implications for hypotheses of the pathogenesis of psychiatric disorders.

Iron Nanowires Coated with Metal–Organic Frameworks for Cell-Type-Specific Multimodal Therapy

Targeted therapy has the potential to revolutionize medical approaches to bring us closer to an effective personalized therapy model. In this work, we developed homologous-targeted magnetic nanowires coated with biologics-loaded zeolitic imidazolate frameworks and cellular membranes (ZNWs) to provide an increased therapeutic potential both in vitro and in vivo. In vitro, the uncoated ZNWs combined with laser treatment reduced cancer cell viability by 34.6%, whereas the coated ZNWs combined with laser treatment reduced cancer cell viability by 45.91%. In vivo, ZNWs accumulated selectively in MCF-7 cancer cells and maintained a high intensity for up to 120 h, which reduced tumor growth significantly. Combining gene and tumor ablation treatment with the homologous targeting ability of ZNWs resulted in about 73% cell death. We believe that using such an assembled coating to camouflage delivery vehicles has effective outcomes in terms of improved targeting and therapeutic efficiency and should be studied further for medical translation.

Multistage-Responsive Gene Editing to Sensitize Ion-Interference Enhanced Carbon Monoxide Gas Therapy.

As a promising therapeutic modality targeting cancer, gas therapy still faces critical challenges, especially in enhancing therapeutic efficacy and avoiding gas poisoning risks. Here, a pH/glutathione (GSH) dual stimuli-responsive CRISPR/Cas9 gene-editing nanoplatform combined with calcium-enhanced CO gas therapy for precise anticancer therapy, is established. In the tumor microenvironment (TME), the fast biodegradation of the CaCO3 layer via pH-induced hydrolyzation allows glucose oxidase (GOx) to catalyze glucose for H2 O2 production, which further reacts with manganese carbonyl (MnCO) and achieves the precise release of CO gas. Simultaneously, in situ Ca2+ overload from CaCO3 degradation disturbs mitochondrial Ca2+ homeostasis, resulting in Ca2+ -driven reactive oxygen species (ROS) formation and subsequent mitochondrial apoptosis signaling pathway activation. Subsequently, by GSH-induced cleavage of a disulfide bond, the released Cas9/sgRNA (RNP) can achieve nuclear factor E2-related factor 2 (Nrf2) gene ablation to sensitize gas therapy by interfering with ROS signaling. This therapeutic modality endows codelivery of CRISPR, ions, and gas with smart control features, which demonstrates great potential for future clinical applications in precise nanomedicine.

Toxic and Phenotypic Effects of AAV_Cre Used to Transduce Mesencephalic Dopaminergic Neurons.

A popular approach to spatiotemporally target genes using the loxP/Cre recombination system is stereotaxic microinjection of adeno-associated virus (AAV) expressing Cre recombinase (AAV_Cre) in specific neuronal structures. Here, we report that AAV_Cre microinjection in the ventral tegmental area (VTA) of ErbB4 Cyt-1-floxed (ErbB4 Cyt-1fl/fl) mice at titers commonly used in the literature (~1012–1013 GC/mL) can have neurotoxic effects on dopaminergic neurons and elicit behavioral abnormalities. However, these effects of AAV_Cre microinjection are independent of ErbB4 Cyt-1 recombination because they are also observed in microinjected wild-type (WT) controls. Mice microinjected with AAV_Cre (1012–1013 GC/mL) exhibit reductions of tyrosine hydroxylase (TH) and dopamine transporter (DAT) expression, loss of dopaminergic neurons, and they behaviorally become hyperactive, fail to habituate in the open field and exhibit sensorimotor gating deficits compared to controls microinjected with AAV_GFP. Importantly, these AAV_Cre non-specific effects are: (1) independent of serotype, (2) occur with vectors expressing either Cre or Cre-GFP fusion protein and (3) preventable by reducing viral titers by 1000-fold (1010 GC/mL), which retains sufficient recombination activity to target floxed genes. Our studies emphasize the importance of including AAV_Cre-injected WT controls in experiments because recombination-independent effects on gene expression, neurotoxicity and behaviors could be erroneously attributed to consequences of gene ablation.

Myc inhibition tips the immune balance to promote antitumor immunity.

Aberrant expression of Myc is one of the most common oncogenic events in human cancers. Scores of Myc inhibitors are currently under development for treating Myc-driven cancers. In addition to directly targeting tumor cells, Myc inhibition has been shown to modulate the tumor microenvironment to promote tumor regression. However, the effect of Myc inhibition on immune cells in the tumor microenvironment remains poorly understood. Here, we show that the adaptive immune system plays a vital role in the antitumor effect of pharmacologic inhibition of Myc. Combining genetic and pharmacologic approaches, we found that Myc inhibition enhanced CD8 T cell function by suppressing the homeostasis of regulatory T (Treg) cells and the differentiation of resting Treg (rTreg) cells to activated Treg (aTreg) cells in tumors. Importantly, we demonstrated that different Myc expression levels confer differential sensitivity of T cell subsets to pharmacologic inhibition of Myc. Although ablation of the Myc gene has been shown to suppress CD8 T cell function, Treg cells, which express much less Myc protein than CD8 T cells, are more sensitive to Myc inhibitors. The differential sensitivity of CD8 T and Treg cells to Myc inhibitors resulted in enhanced CD8 T cell function upon Myc inhibition. Our findings revealed that Myc inhibitors can induce an antitumor immune response during tumor progression.

The mitochondrial NAD kinase functions as a major metabolic regulator upon increased energy demand

ObjectiveThe mitochondrial nicotinamide adenine dinucleotide (NAD) kinase (MNADK) mediates de novo mitochondrial NADP biosynthesis by catalyzing the phosphorylation of NAD to yield NADP. In this study, we investigated the function and mechanistic basis by which MNADK regulates metabolic homeostasis. MethodsGeneralized gene set analysis by aggregating human patient genomic databases, metabolic studies with genetically engineered animal models, mitochondrial bioenergetic analysis, as well as gain- and loss- of-function studies were performed to address the functions and mechanistic basis by which MNADK regulates energy metabolism and redox state associated with metabolic disease. ResultsHuman MNADK common gene variants or decreased expression of the gene are significantly associated with the occurrence of type-2 diabetes, non-alcoholic fatty liver disease (NAFLD), or hepatocellular carcinoma (HCC). Ablation of the MNADK gene in mice led to decreased fat oxidation, coincident with increased respiratory exchange ratio (RER) and decreased energy expenditure upon energy demand triggered by endurance exercise or fasting. On an atherogenic high-fat diet (HFD), MNADK-null mice exhibited hepatic insulin resistance and glucose intolerance, indicating a type-2 diabetes-like phenotype in the absence of MNADK. MNADK deficiency led to a decrease in mitochondrial NADP(H) but an increase in cellular reactive oxygen species (ROS) in mouse livers. Consistently, protein levels of the major metabolic regulators or enzymes were decreased, while their acetylation modifications were increased in the livers of MNADK-null mice. Feeding mice with a HFD caused S-nitrosylation (SNO) modification, a posttranslational modification that represses protein activities, on MNADK protein in the liver. Reconstitution of an SNO-resistant MNADK variant, MNADK-S193, into MNADK-null mice mitigated hepatic steatosis induced by HFD. ConclusionMNADK, the only known mammalian mitochondrial NAD kinase, plays important roles in preserving energy homeostasis to mitigate the risk of metabolic disorders.

Lack of xanthine dehydrogenase leads to a remarkable renal decline in a novel hypouricemic rat model

SummaryUric acid (UA) is the final metabolite in purine catabolism in humans. Previous studies have shown that the dysregulation of UA homeostasis is detrimental to cardiovascular and kidney health. The Xdh gene encodes for the Xanthine Oxidoreductase enzyme group, responsible for producing UA. To explore how hypouricemia can lead to kidney damage, we created a rat model with the genetic ablation of the Xdh gene on the Dahl salt-sensitive rat background (SSXdh−/−). SSXdh−/− rats lacked UA and exhibited impairment in growth and survival. This model showed severe kidney injury with increased interstitial fibrosis, glomerular damage, crystal formation, and an inability to control electrolyte balance. Using a multi-omics approach, we highlighted that lack of Xdh leads to increased oxidative stress, renal cell proliferation, and inflammation. Our data reveal that the absence of Xdh leads to kidney damage and functional decline by the accumulation of purine metabolites in the kidney and increased oxidative stress.

Lower blood glucose and disrupted glucagon signaling in mice with liver-specific ablation of the hepatocyte nuclear factor 4Α (Hnf4A) gene

Background and Aims : Hnf4a gene ablation in mouse liver causes hepatic steatosis, perturbations in HDL structure and function and affects many pathways and genes related to glucose metabolism. Our aim was to investigate the role of liver HNF4A in glucose homeostasis.

Ultra-thin layered double hydroxide-mediated photothermal therapy combine with asynchronous blockade of PD-L1 and NR2F6 inhibit hepatocellular carcinoma

BackgroundThe efficacy of immune checkpoint blockade (ICB), in the treatment of hepatocellular carcinoma (HCC), is limited due to low levels of tumor-infiltrating T lymphocytes and deficient checkpoint blockade in this immunologically "cool" tumor. Thus, combination approaches are needed to increase the response rates of ICB and induce synergistic antitumor immunity.MethodsHerein, we designed a pH-sensitive multifunctional nanoplatform based on layered double hydroxides (LDHs) loaded with siRNA to block the intracellular immune checkpoint NR2F6, together with the asynchronous blockade surface receptor PD-L1 to induce strong synergistic antitumor immunity. Moreover, photothermal therapy (PTT) generated by LDHs after laser irradiation modified an immunologically “cold” microenvironment to potentiate Nr2f6-siRNA and anti-PD-L1 immunotherapy. Flow cytometry was performed to assess the immune responses initiated by the multifunctional nanoplatform.ResultsUnder the slightly acidic tumor extracellular environment, PEG detached and the re-exposed positively charged LDHs enhanced tumor accumulation and cell uptake. The accumulated siRNA suppressed the signal of dual protumor activity in both immune and H22 tumor cells by silencing the NR2F6 gene, which further reduced the tumor burden and enhanced systemic antitumor immunity. The responses include enhanced tumor infiltration by CD4+ helper T cells, CD8+ cytotoxic T cells, and mature dendritic cells; the significantly decreased level of immune suppressed regulator T cells. The therapeutic responses were also attributed to the production of IL-2, IFN-γ, and TNF-α. The prepared nanoparticles also exhibited potential magnetic resonance imaging (MRI) ability, which could serve to guide synergistic immunotherapy treatment.ConclusionsIn summary, the three combinations of PTT, NR2F6 gene ablation and anti-PD-L1 can promote a synergistic immune response to inhibit the progression of primary HCC tumors and prevent metastasis. This study can be considered a proof-of-concept for the targeting of surface and intracellular immune checkpoints to supplement the existing HCC immunotherapy treatments.

Deficient Caveolin-1 Synthesis in Adipocytes Stimulates Systemic Insulin-Independent Glucose Uptake via Extracellular Vesicles.

Caveolin-1 (cav1) is an important structural and signaling component of plasma membrane invaginations called caveolae and is abundant in adipocytes. As previously reported, adipocyte-specific ablation of the cav1 gene (ad-cav1 knockout [KO] mouse) does not result in elimination of the protein, as cav1 protein traffics to adipocytes from neighboring endothelial cells. However, this mouse is a functional KO because adipocyte caveolar structures are depleted. Compared with controls, ad-cav1KO mice on a high-fat diet (HFD) display improved whole-body glucose clearance despite complete loss of glucose-stimulated insulin secretion, blunted insulin-stimulated AKT activation in metabolic tissues, and partial lipodystrophy. The cause is increased insulin-independent glucose uptake by white adipose tissue (AT) and reduced hepatic gluconeogenesis. Furthermore, HFD-fed ad-cav1KO mice display significant AT inflammation, fibrosis, mitochondrial dysfunction, and dysregulated lipid metabolism. The glucose clearance phenotype of the ad-cav1KO mice is at least partially mediated by AT small extracellular vesicles (AT-sEVs). Injection of control mice with AT-sEVs from ad-cav1KO mice phenocopies ad-cav1KO characteristics. Interestingly, AT-sEVs from ad-cav1KO mice propagate the phenotype of the AT to the liver. These data indicate that ad-cav1 is essential for healthy adaptation of the AT to overnutrition and prevents aberrant propagation of negative phenotypes to other organs by EVs.

723 DNA dioxygenases Tet2/3 regulate gene promoter accessibility and three-dimensional chromatin topology in lineage-specific loci to control hair growth

Lineage-specific and functionally-related genes frequently form conserved clusters or loci in the mammalian genomes. Execution of lineage-specific differentiation programs requires tight coordination between many regulators including Ten-eleven translocation (TET) family enzymes catalyzing DNA oxidation. However, the role of Tet genes in the control of cell differentiation at the levels of lineage-specific gene loci is unknown. Epithelial keratin genes are organized in the genome into two distinct (Keratin type I and II) loci on mouse chromosomes 11 and 15. Here, by using Keratin 14-driven ablation of Tet genes in skin epithelial cells, we demonstrate that Tet enzymes play essential roles in the control of gene expression in Keratin type I/II gene loci and execution of hair-specific differentiation program. Mice with Keratin 14-driven ablation of Tet2/Tet3 or all three Tet genes exhibit marked alterations of hair shape and length followed by hair loss. We show that through DNA demethylation, Tet2 and Tet3 control chromatin accessibility, Dlx3 binding and promoter activity of the Krt25 and Krt28 genes regulating hair shape, as well as regulate interactions between the Krt28 gene promoter and distal enhancer. However, Tet2/3 also control three-dimensional chromatin topology in Keratin type I/II gene loci via catalytic activity-independent mechanisms. These data demonstrate for the first time the essential roles for Tet2/3 in establishment of lineage-specific gene expression program and control of Dlx3/Krt25/Krt28 axis in hair follicle epithelial cells and implicate modulation of DNA methylation as a novel approach for hair growth control.