RESUMO
Proper hematopoietic cell fate decisions require co-ordinated functions of transcription factors, their associated co-regulators, and histone-modifying enzymes. Growth factor independence 1 (GFI1) is a zinc finger transcriptional repressor and master regulator of normal and malignant hematopoiesis. While several GFI1-interacting proteins have been described, how GFI1 leverages these relationships to carry out transcriptional repression remains unclear. Here, we describe a functional axis involving GFI1, SMYD2, and LSD1 that is a critical contributor to GFI1-mediated transcriptional repression. SMYD2 methylates lysine-8 (K8) within a -(8)KSKK(11)- motif embedded in the GFI1 SNAG domain. Methylation-defective GFI1 SNAG domain lacks repressor function due to failure of LSD1 recruitment and persistence of promoter H3K4 di-methyl marks. Methylation-defective GFI1 also fails to complement GFI1 depletion phenotypes in developing zebrafish and lacks pro-growth and survival functions in lymphoid leukemia cells. Our data show a discrete methylation event in the GFI1 SNAG domain that facilitates recruitment of LSD1 to enable transcriptional repression and co-ordinate control of hematopoietic cell fate in both normal and malignant settings.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Histona Desmetilases/metabolismo , Fatores de Transcrição/fisiologia , Transcrição Gênica/fisiologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Linhagem da Célula , Metilação de DNA , Proteínas de Ligação a DNA/química , Humanos , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/química , Peixe-ZebraRESUMO
BACKGROUND: Surgical treatment for large-angle exotropia can be challenging. The aim of this study was to evaluate short-term surgical outcomes of patients with large-angle exotropia (≥50Δ) undergoing maximal bilateral lateral rectus muscle recession of 10 mm. METHODS: This was a retrospective study of consecutive patients at our institution who underwent maximal bilateral lateral rectus muscle recession for exodeviation ≥50Δ from January 1, 2008, to July 22, 2022. We subdivided the cohort into large-angle exotropia (largest amount of exodeviation at near and/or distance ≥50Δ and <65Δ) and very large-angle exotropia (largest exodeviation ≥65Δ). Patients with a history of prior eye muscle surgery, neurologic deficits, and three- or four-muscle surgery were excluded. RESULTS: A total of 22 patients were included. Mean preoperative exodeviation at distance was 51.9Δ in the large-angle group and 67.5Δ in the very-large-angle group (P = 0.001). Outcomes for the large-angle and very-large angle groups were, respectively, as follows: mean follow-up, 31.1 weeks and 11.8 weeks (P = 0.97); success, 75.0% and 16.7% (P = 0.02); undercorrection rates, 18.7% and 83.3% (P = 0.01); and mean postoperative exodeviation at distance, 3.7Δ ± 6.3Δ and 28.0Δ ± 13.5Δ (P = 0.001). CONCLUSIONS: Our study identified good surgical outcomes (75%) with maximal bilateral lateral rectus muscle recession of 10 mm in treating patients with large-angle exotropia between 50Δ and <65Δ. Other surgical techniques such as recession-resection and three- or four-muscle surgery may result in better outcomes when treating patients with exotropia ≥65Δ.
Assuntos
Exotropia , Humanos , Exotropia/cirurgia , Seguimentos , Resultado do Tratamento , Estudos Retrospectivos , Visão Binocular/fisiologia , Procedimentos Cirúrgicos Oftalmológicos , Músculos Oculomotores/cirurgiaRESUMO
The liver coordinates the systemic response to nutrient deprivation and availability by producing glucose from gluconeogenesis during fasting and synthesizing lipids via de novo lipogenesis (DNL) when carbohydrates are abundant. Mitochondrial pyruvate metabolism is thought to play important roles in both gluconeogenesis and DNL. We examined the effects of hepatocyte-specific mitochondrial pyruvate carrier (MPC) deletion on the fasting-refeeding response. Rates of DNL during refeeding were impaired by liver MPC deletion, but this did not reduce intrahepatic lipid content. During fasting, glycerol is converted to glucose by two pathways; a direct cytosolic pathway essentially reversing glycolysis and an indirect mitochondrial pathway requiring the MPC. MPC deletion reduced the incorporation of 13C-glycerol into TCA cycle metabolites but not into newly synthesized glucose. However, suppression of glycerol metabolism did not affect glucose concentrations in fasted hepatocyte-specific MPC-deficient mice. Thus, glucose production by kidney and intestine may compensate for MPC deficiency in hepatocytes.
RESUMO
The liver coordinates the systemic response to nutrient deprivation and availability by producing glucose from gluconeogenesis during fasting and synthesizing lipids via de novo lipogenesis (DNL) when carbohydrates are abundant. Mitochondrial pyruvate metabolism is thought to play important roles in both gluconeogenesis and DNL. We examined the effects of hepatocyte-specific mitochondrial pyruvate carrier (MPC) deletion on the fasting-refeeding response. Rates of DNL during refeeding were impaired by hepatocyte MPC deletion, but this did not reduce intrahepatic lipid content. During fasting, glycerol is converted to glucose by two pathways; a direct cytosolic pathway and an indirect mitochondrial pathway requiring the MPC. Hepatocyte MPC deletion reduced the incorporation of 13C-glycerol into TCA cycle metabolites, but not into new glucose. Furthermore, suppression of glycerol and alanine metabolism did not affect glucose concentrations in fasted hepatocyte-specific MPC-deficient mice, suggesting multiple layers of redundancy in glycemic control in mice.
RESUMO
Dysfunctional adipose tissue is believed to promote the development of hepatic steatosis and systemic insulin resistance, but many of the mechanisms involved are still unclear. Lipin 1 catalyzes the conversion of phosphatidic acid to diacylglycerol (DAG), the penultimate step of triglyceride synthesis, which is essential for lipid storage. Herein we found that adipose tissue LPIN1 expression is decreased in people with obesity compared to lean subjects and low LPIN1 expression correlated with multi-tissue insulin resistance and increased rates of hepatic de novo lipogenesis. Comprehensive metabolic and multi-omic phenotyping demonstrated that adipocyte-specific Lpin1-/- mice had a metabolically-unhealthy phenotype, including liver and skeletal muscle insulin resistance, hepatic steatosis, increased hepatic de novo lipogenesis, and transcriptomic signatures of nonalcoholic steatohepatitis that was exacerbated by high-fat diets. We conclude that adipocyte lipin 1-mediated lipid storage is vital for preserving adipose tissue and systemic metabolic health and its loss predisposes mice to nonalcoholic steatohepatitis.
RESUMO
OBJECTIVE: Monoacylglycerol O-acyltransferase 1 (Mogat1), a lipogenic enzyme that converts monoacylglycerol to diacylglycerol, is highly expressed in adipocytes and may regulate lipolysis by re-esterifying fatty acids released during times when lipolytic rates are low. However, the role of Mogat1 in regulating adipocyte fat storage during differentiation and diet-induced obesity is relatively understudied. METHODS: Here, adipocyte-specific Mogat1 knockout mice were generated and subjected to a high-fat diet to determine the effects of Mogat1 deficiency on diet-induced obesity. Mogat1 floxed mice were also used to develop preadipocyte cell lines wherein Mogat1 could be conditionally knocked out to study adipocyte differentiation in vitro. RESULTS: In preadipocytes, it was found that Mogat1 knockout at the onset of preadipocyte differentiation prevented the accumulation of glycerolipids and reduced the differentiation capacity of preadipocytes. However, the loss of adipocyte Mogat1 did not affect weight gain or fat mass induced by a high-fat diet in mice. Furthermore, loss of Mogat1 in adipocytes did not affect plasma lipid or glucose concentrations or insulin tolerance. CONCLUSIONS: These data suggest Mogat1 may play a role in adipocyte differentiation in vitro but not adipose tissue expansion in response to nutrient overload in mice.
Assuntos
Adiposidade , Monoglicerídeos , Camundongos , Animais , Monoglicerídeos/metabolismo , Obesidade/metabolismo , Adipócitos/metabolismo , Dieta Hiperlipídica , Diferenciação Celular , Camundongos Knockout , Aciltransferases/metabolismo , Camundongos Endogâmicos C57BLRESUMO
Hepatic gluconeogenesis from amino acids contributes significantly to diabetic hyperglycemia, but the molecular mechanisms involved are incompletely understood. Alanine transaminases (ALT1 and ALT2) catalyze the interconversion of alanine and pyruvate, which is required for gluconeogenesis from alanine. We find that ALT2 is overexpressed in the liver of diet-induced obese and db/db mice and that the expression of the gene encoding ALT2 (GPT2) is downregulated following bariatric surgery in people with obesity. The increased hepatic expression of Gpt2 in db/db liver is mediated by activating transcription factor 4, an endoplasmic reticulum stress-activated transcription factor. Hepatocyte-specific knockout of Gpt2 attenuates incorporation of 13C-alanine into newly synthesized glucose by hepatocytes. In vivo Gpt2 knockdown or knockout in liver has no effect on glucose concentrations in lean mice, but Gpt2 suppression alleviates hyperglycemia in db/db mice. These data suggest that ALT2 plays a significant role in hepatic gluconeogenesis from amino acids in diabetes.
Assuntos
Diabetes Mellitus , Hiperglicemia , Alanina/farmacologia , Alanina Transaminase/metabolismo , Aminoácidos/metabolismo , Animais , Diabetes Mellitus/metabolismo , Gluconeogênese , Glucose/metabolismo , Humanos , Hiperglicemia/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Obesidade/metabolismoRESUMO
OBJECTIVE: Monoacylglycerol acyltransferase (MGAT) enzymes catalyze the synthesis of diacylglycerol from monoacylglycerol. Previous work has suggested the importance of MGAT activity in the development of obesity-related hepatic insulin resistance. Indeed, antisense oligonucleotide (ASO)-mediated knockdown of Mogat1 mRNA, which encodes MGAT1, reduced hepatic MGAT activity and improved glucose tolerance and insulin resistance in high-fat diet (HFD)-fed mice. However, recent work has suggested that some ASOs may have off-target effects on body weight and metabolic parameters via activation of the interferon alpha/beta receptor 1 (IFNAR-1) pathway. METHODS: Mice with whole-body Mogat1 knockout or a floxed allele for Mogat1 to allow for liver-specific Mogat1-knockout (by either a liver-specific transgenic or adeno-associated virus-driven Cre recombinase) were generated. These mice were placed on an HFD, and glucose metabolism and insulin sensitivity were assessed after 16 weeks on diet. In some experiments, mice were treated with control scramble or Mogat1 ASOs in the presence or absence of IFNAR-1 neutralizing antibody. RESULTS: Genetic deletion of hepatic Mogat1, either acutely or chronically, did not improve hepatic steatosis, glucose tolerance, or insulin sensitivity in HFD-fed mice. Furthermore, constitutive Mogat1 knockout in all tissues actually exacerbated HFD-induced obesity, insulin sensitivity, and glucose intolerance on an HFD. Despite markedly reduced Mogat1 expression, liver MGAT activity was unaffected in all knockout mouse models. Mogat1 overexpression in hepatocytes increased liver MGAT activity and TAG content in low-fat-fed mice but did not cause insulin resistance. Multiple Mogat1 ASO sequences improved glucose tolerance in both wild-type and Mogat1 null mice, suggesting an off-target effect. Hepatic IFNAR-1 signaling was activated by multiple Mogat1 ASOs, but its blockade did not prevent the effects of either Mogat1 ASO on glucose homeostasis. CONCLUSION: These results indicate that genetic loss of Mogat1 does not affect hepatic MGAT activity or metabolic homeostasis on HFD and show that multiple Mogat1 ASOs improve glucose metabolism through effects independent of targeting Mogat1 or activation of IFNAR-1 signaling.
Assuntos
Aciltransferases/genética , Aciltransferases/metabolismo , Metabolismo dos Carboidratos , Oligonucleotídeos Antissenso/metabolismo , Animais , Dieta Hiperlipídica , Diglicerídeos/metabolismo , Fígado Gorduroso/metabolismo , Feminino , Glucose/metabolismo , Intolerância à Glucose/metabolismo , Resistência à Insulina , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/metabolismo , Oligonucleotídeos Antissenso/genética , Fenótipo , Receptor de Interferon alfa e beta/metabolismo , TranscriptomaRESUMO
Growth factor independence 1B (GFI1B) coordinates assembly of transcriptional repressor complexes comprised of corepressors and histone-modifying enzymes to control gene expression programs governing lineage allocation in hematopoiesis. Enforced expression of GFI1B in K562 erythroleukemia cells favors erythroid over megakaryocytic differentiation, providing a platform to define molecular determinants of binary fate decisions triggered by GFI1B. We deployed proteome-wide proximity labeling to identify factors whose inclusion in GFI1B complexes depends upon GFI1B's obligate effector, lysine-specific demethylase 1 (LSD1). We show that GFI1B preferentially recruits core and putative elements of the BRAF-histone deacetylase (HDAC) (BHC) chromatin-remodeling complex (LSD1, RCOR1, HMG20A, HMG20B, HDAC1, HDAC2, PHF21A, GSE1, ZMYM2, and ZNF217) in an LSD1-dependent manner to control acquisition of erythroid traits by K562 cells. Among these elements, depletion of both HMG20A and HMG20B or of GSE1 blocks GFI1B-mediated erythroid differentiation, phenocopying impaired differentiation brought on by LSD1 depletion or disruption of GFI1B-LSD1 binding. These findings demonstrate the central role of the GFI1B-LSD1 interaction as a determinant of BHC complex recruitment to enable cell fate decisions driven by GFI1B.
Assuntos
Células Eritroides/citologia , Histona Desmetilases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/metabolismo , Animais , Células COS , Diferenciação Celular , Chlorocebus aethiops , Regulação para Baixo , Células Eritroides/metabolismo , Histona Desacetilases/metabolismo , Humanos , Células K562 , Fenótipo , Acetato de Tetradecanoilforbol/farmacologia , Transcrição GênicaRESUMO
INTRODUCTION: The etiology, frequency, manifestation, and treatment of dry eye syndrome are commonly influenced by sex and gender. MATERIALS AND METHODS: This study aims to review the differences in epidemiology, pathophysiology, and associated diseases between the sexes. The terms men and male and women and female are used interchangeably throughout the review to refer to biological sex. RESULTS: There are numerous objective and subjective markers of dry eye syndrome but not one diagnostic criterion. There are numerous associated conditions with dry eye syndrome varying from autoimmune to allergic. Large epidemiologic studies reviewed suggest that there does indeed exist a difference between dry eye symptoms between men and women, with women having dry eye signs and reporting dry eye symptoms more often than men. The increased prevalence in women could be correlated to an increased association with certain systemic diseases, specifically autoimmune diseases, and to hormonal variations. Several studies found equivocal data about prevalence of dry eye symptoms between men and women. DISCUSSION: Interpreting studies that investigate epidemiology, pathogenesis, and treatment of dry-eye conditions is complicated by the lack of universally adapted diagnostic criteria and standardized, specific diagnostic tests, and inter-study variability in the definition of dry eye syndrome.
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Síndromes do Olho Seco , Disparidades nos Níveis de Saúde , Síndromes do Olho Seco/diagnóstico , Síndromes do Olho Seco/epidemiologia , Síndromes do Olho Seco/fisiopatologia , Feminino , Humanos , Masculino , Prevalência , Fatores SexuaisRESUMO
Cell fate specification requires precise coordination of transcription factors and their regulators to achieve fidelity and flexibility in lineage allocation. The transcriptional repressor growth factor independence 1 (GFI1) is comprised of conserved Snail/Slug/Gfi1 (SNAG) and zinc finger motifs separated by a linker region poorly conserved with GFI1B, its closest homolog. Moreover, GFI1 and GFI1B coordinate distinct developmental fates in hematopoiesis, suggesting that their functional differences may derive from structures within their linkers. We show a binding interface between the GFI1 linker and the SP-RING domain of PIAS3, an E3-SUMO (small ubiquitin-related modifier) ligase. The PIAS3 binding region in GFI1 contains a conserved type I SUMOylation consensus element, centered on lysine-239 (K239). In silico prediction algorithms identify K239 as the only high-probability site for SUMO modification. We show that GFI1 is modified by SUMO at K239. SUMOylation-resistant derivatives of GFI1 fail to complement Gfi1 depletion phenotypes in zebrafish primitive erythropoiesis and granulocytic differentiation in cultured human cells. LSD1/CoREST recruitment and MYC repression by GFI1 are profoundly impaired for SUMOylation-resistant GFI1 derivatives, while enforced expression of MYC blocks granulocytic differentiation. These findings suggest that SUMOylation within the GFI1 linker favors LSD1/CoREST recruitment and MYC repression to govern hematopoietic differentiation.
Assuntos
Hematopoese , Histona Desmetilases/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Inibidoras de STAT Ativados/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/metabolismo , Animais , Sítios de Ligação , Células COS , Diferenciação Celular , Chlorocebus aethiops , Regulação da Expressão Gênica , Células HEK293 , Células HL-60 , Humanos , Lisina/metabolismo , Camundongos , Chaperonas Moleculares/química , Células NIH 3T3 , Ligação Proteica , Proteínas Inibidoras de STAT Ativados/química , Proteínas Proto-Oncogênicas/química , Proteínas Repressoras/química , SumoilaçãoRESUMO
Isolation of ventricular cardiomyocytes (vCMs) has been challenging due to the lack of specific surface markers. Here we show that vCMs can be purified from differentiating mouse embryonic stem cells (mESCs) using molecular beacons (MBs) targeting specific intracellular mRNAs. We designed MBs (IRX4 MBs) to target mRNA encoding Iroquois homeobox protein 4 (Irx4), a transcription factor specific for vCMs. To purify mESC vCMs, IRX4 MBs were delivered into cardiomyogenically differentiating mESCs, and IRX4 MBs-positive cells were FACS-sorted. We found that, of the cells isolated, ~98% displayed vCM-like action potentials by electrophysiological analyses. These MB-purified vCMs continuously maintained their CM characteristics as verified by spontaneous beating, Ca(2+) transient, and expression of vCM-specific proteins. Our study shows the feasibility of isolating pure vCMs via cell sorting without modifying host genes. The homogeneous and functional ventricular CMs generated via the MB-based method can be useful for disease investigation, drug discovery, and cell-based therapies.
Assuntos
Separação Celular/métodos , Células-Tronco Embrionárias/citologia , Ventrículos do Coração/citologia , Proteínas de Homeodomínio/genética , Miócitos Cardíacos/citologia , Potenciais de Ação , Animais , Sequência de Bases , Diferenciação Celular , Células Cultivadas , Citometria de Fluxo , Camundongos , Sondas de Oligonucleotídeos/genética , RNA Mensageiro/genéticaRESUMO
AIMS: To evaluate the ability of N-acetylglucosamine (GlcNAc) decorated nanoparticles and their cargo to modulate calcium handling in failing cardiac myocytes (CMs). MATERIALS & METHODS: Primary CMs isolated from normal and failing hearts were treated with GlcNAc nanoparticles in order to assess the ability of the nanoparticles and their cargo to correct dysfunctional calcium handling in failing myocytes. RESULTS & CONCLUSION: GlcNAc particles reduced aberrant calcium release in failing CMs and restored sarcomere function. Additionally, encapsulation of a small calcium-modulating protein, S100A1, in GlcNAc nanoparticles also showed improved calcium regulation. Thus, the development of our bioactive nanoparticle allows for a 'two-hit' treatment, by which the cargo and also the nanoparticle itself can modulate intracellular protein activity.
Assuntos
Acetilglucosamina/administração & dosagem , Insuficiência Cardíaca/tratamento farmacológico , Miócitos Cardíacos/metabolismo , Nanopartículas/administração & dosagem , Acetilglucosamina/química , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Insuficiência Cardíaca/metabolismo , Humanos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Nanopartículas/química , Proteínas S100/metabolismo , Sarcômeros/metabolismo , Sarcômeros/patologiaRESUMO
The evolutionarily conserved Mdm20 protein (Mdm20p) plays an important role in tropomyosin-F-actin interactions that generate actin filaments and cables in budding yeast. However, Mdm20p is not a structural component of actin filaments or cables, and its exact function in cable stability has remained a mystery. Here, we show that cells lacking Mdm20p fail to N-terminally acetylate Tpm1p, an abundant form of tropomyosin that binds and stabilizes actin filaments and cables. The F-actin-binding activity of unacetylated Tpm1p is reduced severely relative to the acetylated form. These results are complemented by the recent report that Mdm20p copurifies with one of three acetyltransferases in yeast, the NatB complex. We present genetic evidence that Mdm20p functions cooperatively with Nat3p, the catalytic subunit of the NatB acetyltransferase. These combined results strongly suggest that Mdm20p-dependent, N-terminal acetylation of Tpm1p by the NatB complex is required for Tpm1p association with, and stabilization of, actin filaments and cables.