ABSTRACT
CRISPR-mediated gene activation (CRISPRa) is a promising therapeutic gene editing strategy without inducing DNA double-strand breaks (DSBs). However, in vivo implementation of these CRISPRa systems remains a challenge. Here, we report a compact and robust miniCas9 activator (termed miniCAFE) for in vivo activation of endogenous target genes. The system relies on recruitment of an engineered minimal nuclease-null Cas9 from Campylobacter jejuni and potent transcriptional activators to a target locus by a single guide RNA. It enables robust gene activation in human cells even with a single DNA copy and is able to promote lifespan of Caenorhabditis elegans through activation of longevity-regulating genes. As proof-of-concept, delivered within an all-in-one adeno-associated virus (AAV), miniCAFE can activate Fgf21 expression in the liver and regulate energy metabolism in adult mice. Thus, miniCAFE holds great therapeutic potential against human diseases.
Subject(s)
CRISPR-Associated Protein 9/metabolism , CRISPR-Cas Systems , Fibroblast Growth Factors/metabolism , Gene Editing , RNA, Guide, Kinetoplastida/metabolism , Animals , Caenorhabditis elegans , Campylobacter jejuni , HEK293 Cells , Humans , Melanoma, Experimental , Mice , Mice, Inbred C57BLABSTRACT
Disregulation of fatty acid oxidation, one of the major mechanisms for maintaining hepatic lipid homeostasis under fasting conditions, leads to hepatic steatosis. Although obesity and type 2 diabetes-induced endoplasmic reticulum (ER) stress contribute to hepatic steatosis, it is largely unknown how ER stress regulates fatty acid oxidation. Here we show that fasting glucagon stimulates the dephosphorylation and nuclear translocation of histone deacetylase 5 (HDAC5), where it interacts with PPARα and promotes transcriptional activity of PPARα. As a result, overexpression of HDAC5 but not PPARα binding-deficient HDAC5 in liver improves lipid homeostasis, whereas RNAi-mediated knockdown of HDAC5 deteriorates hepatic steatosis. ER stress inhibits fatty acid oxidation gene expression via calcium/calmodulin-dependent protein kinase II-mediated phosphorylation of HDAC5. Most important, hepatic overexpression of a phosphorylation-deficient mutant HDAC5 2SA promotes hepatic fatty acid oxidation gene expression and protects against hepatic steatosis in mice fed a high-fat diet. We have identified HDAC5 as a novel mediator of hepatic fatty acid oxidation by fasting and ER stress signals, and strategies to promote HDAC5 dephosphorylation could serve as new tools for the treatment of obesity-associated hepatic steatosis.
Subject(s)
Endoplasmic Reticulum Stress , Fasting/metabolism , Fatty Acids/metabolism , Histone Deacetylases/metabolism , Liver/metabolism , Animals , Male , Mice , Mice, Inbred C57BL , Oxidation-ReductionABSTRACT
The overall survival of patients with cervical cancer has improved due to detection at an early stage and availability of comprehensive treatments in China. As patients' lives prolonged, it is important to understand their health-related quality of life (QoL) during and after treatment. We used the EQ-5D questionnaire to assess QoL of 194 patients with cervical lesions at Sichuan University West China Second Hospital between May 2010 and January 2011. Patients were surveyed before primary treatment and at 1, 3, and 6 months after primary treatment. Results showed a consistent decline in EQ-5D scores in the spectrum of cervical lesions at each time point after treatment (all P < 0.05). For patients with precursor lesions, there was an increasing trend along the timeline of treatment (P < 0.01). For patients with early-stage cervical cancer, EQ-5D scores declined in the first month (P = 0.01) and gradually increased to higher levels at 6 months post-treatment than those before treatment (P < 0.01). EQ-5D scores followed a similar trend in patients with advanced cervical cancer (P = 0.04), though they did not statistically rebound after 6 months (0.84 ± 0.19 vs. 0.86 ± 0.11, P = 0.62). Regarding advanced cervical cancer, EQ-5D scores for women above 40 years of age appeared to recover more rapidly and reached higher levels than those for women below 40 years (P = 0.03). Caution and extra care are recommended in the early period of cervical cancer treatment given the slight deterioration in the QoL, and in particular, for younger cervical cancer patients. Our study implies that health care providers may need to improve the health-related QoL of cervical cancer patients.
Subject(s)
Age Factors , Quality of Life , Uterine Cervical Dysplasia , Uterine Cervical Neoplasms , China , Female , Humans , Neoplasm Staging , Prospective Studies , Surveys and QuestionnairesABSTRACT
The imbalance between energy intake and energy expenditure leads to the prevalence of obesity worldwide. A strategy to simultaneously limit energy intake and promote energy expenditure would be an important new obesity treatment. Here, we identified rhamnose as a nonnutritive sweetener to promote adipose thermogenesis and energy expenditure. Rhamnose promotes cAMP production and PKA activation through dopamine receptor D1 in adipose tissue. As a result, rhamnose administration promotes UCP1-dependent thermogenesis and ameliorates obesity in mice. Thus, we have demonstrated a rhamnose-dopamine receptor D1-PKA axis critical for thermogenesis, and that rhamnose may have a role in therapeutic molecular diets against obesity.
Subject(s)
Adipose Tissue, Brown , Rhamnose , Mice , Animals , Rhamnose/metabolism , Adipose Tissue, Brown/metabolism , Obesity/metabolism , Energy Metabolism/physiology , Receptors, Dopamine/metabolism , Thermogenesis/physiology , Uncoupling Protein 1/genetics , Uncoupling Protein 1/metabolism , Adipose Tissue, White/metabolism , Mice, Inbred C57BLABSTRACT
Hepatic endoplasmic reticulum (ER) stress is a hallmark of obesity-induced liver steatosis and contributes to the progress of steatosis and insulin resistance in liver. However, its influence on adipose function is still unclear. Here, we identify a hepatic ER stress-induced activating transcription factor 4 (ATF4)-cold-inducible RNA-binding protein (CIRP)-angiopoietin-related protein3 (ANGPTL3) cascade critical for the regulation of adipose browning. We find that obesity increases CIRP expression in liver through ER stress-induced ATF4. CIRP in turn binds to the 3' UTR and increases mRNA stability of ANGPTL3. ANGPTL3 secreted from liver suppresses uncoupling protein 1 expression through integrin αvß3 and c-Jun N-terminal kinase in adipose tissue. While hepatic expression of either ATF4, CIRP, or ANGPTL3 suppresses adipose browning, knockdown of CIRP and ANGPTL3 in liver or administration of integrin αvß3 inhibitor cilengitide increases adipose browning process. Taken together, we identify a communication mechanism to link hepatic ER stress and adipose browning that may imply a reciprocal regulation of obesity and liver steatosis.
Subject(s)
Activating Transcription Factor 4 , Fatty Liver , 3' Untranslated Regions , Activating Transcription Factor 4/metabolism , Adipose Tissue/metabolism , Angiopoietin-Like Protein 3 , Angiopoietin-like Proteins/genetics , Angiopoietin-like Proteins/metabolism , Angiopoietins/genetics , Angiopoietins/metabolism , Endoplasmic Reticulum Stress/genetics , Fatty Liver/metabolism , Humans , Integrins/metabolism , JNK Mitogen-Activated Protein Kinases/metabolism , Liver/metabolism , Obesity/metabolism , RNA-Binding Proteins/metabolism , Uncoupling Protein 1/metabolismABSTRACT
The present study aimed to identify the diseasecausing gene of a fourgeneration Chinese family affected with congenital posterior subcapsular cataracts (CPSC), to additionally investigate the frequency of paired like homeodomain 3 (PITX3) mutations in Chinese patients with autosomal dominant congenital cataract (ADCC) and to analyze the pathogenesis of the mutations identified in the present study. Whole exome sequencing (WES) was utilized to identify the genetic cause of CPSC in the fourgeneration family. Sanger sequencing was performed to verify the WES results and to screen for mutations of the PITX3 gene in probands of an additional 194 Chinese ADCC families. Cosegregation analysis was performed in the family members with available DNA. Subcellular localization analyses and transactivation assays were performed for the PITX3 mutations identified. From the WES data, the c.608delC (p.A203GfsX106) mutation of PITX3 was identified in the fourgeneration family with CPSC. A second PITX3 mutation c.640_656del (p.A214RfsX42) was detected in two of the additional 194 ADCC families and one of these two families exhibited incomplete penetrance. Functional studies indicated that these 2 PITX3 mutant proteins retained a nuclear localization pattern, but resulted in decreased transactivation activity, similar to other previously identified PITX3 mutations. In the present study, 2 different mutations (p.A203GfsX106 and p.A214RfsX42) in PITX3 were identified as the causative defect in a fourgeneration family with CPSC and two ADCC families, respectively. The prevalence of PITX3 geneassociated cataract was 1.54% (3/195) in the Chinese congenital cataract (CC) family cohort. In vitro functional analyses of these 2 PITX3 mutations were performed, in order to enhance understanding of the pathogenesis of CC caused by PITX3 mutations.
Subject(s)
Asian People/genetics , Cataract/genetics , Genetic Association Studies , Genetic Predisposition to Disease , Homeodomain Proteins/genetics , Mutation , Transcription Factors/genetics , Cataract/epidemiology , Computational Biology/methods , Female , Fluorescent Antibody Technique , Gene Expression Regulation , Homeodomain Proteins/metabolism , Humans , Intracellular Space/metabolism , Male , Protein Binding , Protein Transport , Transcription Factors/metabolism , Exome SequencingABSTRACT
Hormonal signals help to maintain glucose and lipid homeostasis in the liver during the periods of fasting. Glucagon, a pancreas-derived hormone induced by fasting, promotes gluconeogenesis through induction of intracellular cAMP production. Glucagon also stimulates hepatic fatty acid oxidation but the underlying mechanism is poorly characterized. Here we report that following the acute induction of gluconeogenic genes Glucose 6 phosphatase (G6Pase) and Phosphoenolpyruvate carboxykinase (Pepck) expression through cAMP-response element-binding protein (CREB), glucagon triggers a second delayed phase of fatty acid oxidation genes Acyl-coenzyme A oxidase (Aox) and Carnitine palmitoyltransferase 1a (Cpt1a) expression via extracellular cAMP. Increase in extracellular cAMP promotes PPARα activity through direct phosphorylation by AMP-activated protein kinase (AMPK), while inhibition of cAMP efflux greatly attenuates Aox and Cpt1a expression. Importantly, cAMP injection improves lipid homeostasis in fasted mice and obese mice, while inhibition of cAMP efflux deteriorates hepatic steatosis in fasted mice. Collectively, our results demonstrate the vital role of glucagon-stimulated extracellular cAMP in the regulation of hepatic lipid metabolism through AMPK-mediated PPARα activation. Therefore, strategies to improve cAMP efflux could serve as potential new tools to prevent obesity-associated hepatic steatosis.
Subject(s)
Cyclic AMP/metabolism , Glucagon/pharmacology , Hepatocytes/drug effects , Hepatocytes/metabolism , Lipid Metabolism/drug effects , Animals , Cells, Cultured , Fatty Acids/metabolism , Fatty Liver/drug therapy , Fatty Liver/etiology , Gene Expression Regulation/physiology , Lipid Metabolism/physiology , Mice , Multidrug Resistance-Associated Proteins/genetics , Multidrug Resistance-Associated Proteins/metabolism , Obesity/complications , Oxidation-ReductionABSTRACT
The NF-κB pathway has important roles in innate immune responses and its regulation is critical to maintain immune homeostasis. Here, we report a newly discovered feedback mechanism for the regulation of this pathway by TLR ligands in macrophages. Lipopolysaccharide (LPS) induced the expression of ICER via p38-mediated activation of CREB in macrophages. ICER, in turn, inhibited the transcriptional activity of NF-κB by direct interaction with the p65 subunit of NF-κB. Deficiency in ICER elevated binding of NF-κB to promoters of pro-inflammatory genes and their subsequent gene expression. Mice deficient in ICER were hypersensitive to LPS-induced endotoxic shock and showed propagated inflammation. Whereas ICER expression in ICER KO bone marrow transplanted mice rescued the ultra-inflammation phenotype, expression of a p65 binding-deficient ICER mutant failed to do so. Our results thus establish p38-CREB-ICER as key components of a negative feedback mechanism necessary to regulate TLR-driven inflammation.
Subject(s)
Cyclic AMP Response Element Modulator/genetics , Immunity, Innate , NF-kappa B/metabolism , Toll-Like Receptors/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism , Animals , Bone Marrow Transplantation , Cells, Cultured , Cyclic AMP Response Element Modulator/deficiency , Cyclic AMP Response Element Modulator/metabolism , Inflammation/metabolism , Inflammation/pathology , Interleukin-6/blood , Interleukin-6/metabolism , Lipopolysaccharides/toxicity , Liver/metabolism , Macrophages, Peritoneal/cytology , Macrophages, Peritoneal/drug effects , Macrophages, Peritoneal/metabolism , Male , Mice , Mice, Inbred C57BL , Promoter Regions, Genetic , Signal Transduction/drug effects , Spleen/metabolism , Transcription Factor RelA/metabolism , Tumor Necrosis Factor-alpha/blood , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Although hypoglycemia has been documented as a major cause of high mortality in the setting of septic shock, the mechanism of hypoglycemia in infection has not been clearly determined. Hepatic gluconeogenesis serves as an important mechanism to maintain glucose levels under physiological conditions and CREB coactivator CRTC2 plays an important role in regulating gluconeogenic gene expression. Here, we show that triggering of the Toll-like receptor 4 pathway in response to endotoxin lipopolysaccharide (LPS) inhibits gluconeogenic gene expression and hepatic glucose output by blocking CRTC2 activation. Interleukin-1ß (IL-1ß) is found to disrupt gluconeogenic gene expression via the activation of the E3 ubiquitin ligase TRAF6, a key component of the Toll-like receptor 4 signaling pathway that associates with and ubiquitinates CRTC2. TRAF6 promotes the K63-linked ubiquitination of CRTC2, a modification that blocks binding of calcineurin at an adjacent calcineurin-binding site, thereby disrupting CRTC2 dephosphorylation in response to glucagon signals. Mutation of TRAF6-binding sites or ubiquitination site in CRTC2 rescues hepatic gluconeogenesis in LPS-challenged mice. These results suggest that pro-inflammatory signals intersect with the CRTC2 pathway in liver, thus contributing to hypoglycemia caused by infection.
ABSTRACT
Fatty acid oxidation and subsequent ketogenesis is one of the major mechanisms to maintain hepatic lipid homeostasis under fasting conditions. Fasting hormone glucagon has been shown to stimulate ketone body production through activation of PPARα; however, the signal pathway linking glucagon to PPARα is largely undiscovered. Here we report that a SIK2-p300-PPARα cascade mediates glucagon's effect on ketogenesis. p300 interacts with PPARα through a conserved LXXLL motif and enhances its transcriptional activity. SIK2 disrupts p300-PPARα interaction by direct phosphorylation of p300 at Ser89, which in turn decreases PPARα-mediated ketogenic gene expression. Moreover, SIK2 phosphorylation defective p300 (p300 S89A) shows increased interaction with PPARα and abolishes suppression of SIK2 on PPARα-mediated ketogenic gene expression in liver. Taken together, our results unveil the signal pathway that mediates fasting induced ketogenesis to maintain hepatic lipid homeostasis.
Subject(s)
E1A-Associated p300 Protein/metabolism , Lipid Metabolism/physiology , PPAR alpha/metabolism , Protein Serine-Threonine Kinases/metabolism , Amino Acid Motifs , Animals , E1A-Associated p300 Protein/chemistry , E1A-Associated p300 Protein/genetics , Fasting , Genes, Reporter , Hep G2 Cells , Humans , Immunoblotting , Liver/metabolism , Male , Mice , Mice, Inbred C57BL , Phosphorylation , Protein Binding , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/genetics , RNA Interference , RNA, Messenger/metabolism , RNA, Small Interfering/metabolismABSTRACT
OBJECTIVE: Gastric cancer remains a major health problem in China. We hypothesized that XRCC1 Arg194Trp and ADPRT Val762Ala may be associated with risk. METHODS: We designed a multicenter 1:1 matched case- control study of 307 pairs of gastric cancers and controls between October 2010 and August 2011. XRCC1 Arg194Trp and ADPRT Val762Ala were sequenced, and demographic data as well as lifestyle factors were collected using a self-designed questionnaire. RESULTS: Individuals carrying XRCC1 Trp/Trp or Arg/Trp variant genotype had a significantly increased risk of gastric cancer (OR, 1.718; 95% CI, 1.190-2.479), while the OR for ADPRT Val762Ala variant genotype (Ala/Ala or Val/Ala) was 1.175 (95% CI, 0.796-1.737). No gene-gene or gene-environment interactions were found. In addition, family history of cancer and drinkers proportion were higher among cases than among controls (P<0.05). CONCLUSIONS: XRCC1 194 Arg/Trp or Trp/Trp genotype, family history of cancer, and drinking are suspected risk factors of gastric cancer from our study. Our findings may offer insight into further similar large gene-environment and gene-gene studies in this region.