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Metabolic dysfunction-associated steatotic liver disease (MASLD) is a growing cause of morbidity with limited treatment options. Thus, accurate in vitro systems to test new therapies are indispensable. While recently, human liver organoid models have emerged to assess steatotic liver disease, a systematic evaluation of their translational potential is still missing. Here, we evaluated human liver organoid models of MASLD, comparatively testing disease induction in three conditions: oleic acid, palmitic acid, and TGF-ß1. Through single-cell analyses, we find that all three models induce inflammatory signatures, but only TGF-ß1 promotes collagen production, fibrosis, and hepatic stellate cell expansion. In striking contrast, oleic acid ameliorates fibrotic signatures and reduces the hepatic stellate cell population. Linking data from each model to gene expression signatures associated with MASLD disease progression further demonstrates that palmitic acid and TGF-ß1 more robustly model inflammation and fibrosis. Our findings highlight the importance of stratifying MASLD organoid models by signatures of clinical disease progression, provide a single-cell reference to benchmark future organoid injury models, and allow us to study evolving steatohepatitis, fibrosis, and HSC susceptibility to injury in a dynamic, multi-lineage human in vitro system.
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Hígado Graso , Cirrosis Hepática , Humanos , Cirrosis Hepática/genética , Cirrosis Hepática/metabolismo , Factor de Crecimiento Transformador beta1/genética , Factor de Crecimiento Transformador beta1/metabolismo , Hígado Graso/genética , Perfilación de la Expresión Génica , Progresión de la EnfermedadRESUMEN
Transforming growth factor beta (TGF-ß) signaling, mediated through the transcription factors Smad2 and Smad3 (Smad2/3), directs different responses in different cell types. Here we report that Smad3 co-occupies the genome with cell-type-specific master transcription factors. Thus, Smad3 occupies the genome with Oct4 in embryonic stem cells (ESCs), Myod1 in myotubes, and PU.1 in pro-B cells. We find that these master transcription factors are required for Smad3 occupancy and that TGF-ß signaling largely affects the genes bound by the master transcription factors. Furthermore, we show that induction of Myod1 in nonmuscle cells is sufficient to redirect Smad3 to Myod1 sites. We conclude that cell-type-specific master transcription factors determine the genes bound by Smad2/3 and are thus responsible for orchestrating the cell-type-specific effects of TGF-ß signaling.
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Transducción de Señal , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Diferenciación Celular , Células Madre Embrionarias , Elementos de Facilitación Genéticos , Humanos , Ratones , Proteína MioD/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Proteína smad3/metabolismoRESUMEN
BACKGROUND AND AIMS: Fibrosis is the common end point for all forms of chronic liver injury, and the progression of fibrosis leads to the development of end-stage liver disease. Activation of HSCs and their transdifferentiation into myofibroblasts results in the accumulation of extracellular matrix proteins that form the fibrotic scar. Long noncoding RNAs regulate the activity of HSCs and provide targets for fibrotic therapies. APPROACH AND RESULTS: We identified long noncoding RNA TILAM located near COL1A1 , expressed in HSCs, and induced with liver fibrosis in humans and mice. Loss-of-function studies in human HSCs and human liver organoids revealed that TILAM regulates the expression of COL1A1 and other extracellular matrix genes. To determine the role of TILAM in vivo, we annotated the mouse ortholog ( Tilam ), generated Tilam- deficient green fluorescent protein-reporter mice, and challenged these mice in 2 different models of liver fibrosis. Single-cell data and analysis of single-data and analysis of Tilam-deficient reporter mice revealed that Tilam is induced in murine HSCs with the development of fibrosis in vivo. Tilam -deficient reporter mice revealed that Tilam is induced in murine HSCs with the development of fibrosis in vivo. Furthermore, loss of Tilam expression attenuated the development of fibrosis in the setting of in vivo liver injury. Finally, we found that TILAM interacts with promyelocytic leukemia nuclear body scaffold protein to regulate a feedback loop by which TGF-ß2 reinforces TILAM expression and nuclear localization of promyelocytic leukemia nuclear body scaffold protein to promote the fibrotic activity of HSCs. CONCLUSIONS: TILAM is activated in HSCs with liver injury and interacts with promyelocytic leukemia nuclear body scaffold protein to drive the development of fibrosis. Depletion of TILAM may serve as a therapeutic approach to combat the development of end-stage liver disease.
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The human immunodeficiency virus type 1 (HIV-1) encodes multiple RNA molecules. Transcripts that originate from the proviral 5' long terminal repeat (LTR) function as messenger RNAs for the expression of 16 different mature viral proteins. In addition, HIV-1 expresses an antisense transcript (Ast) from the 3'LTR, which has both protein-coding and noncoding properties. While the mechanisms that regulate the coding and noncoding activities of Ast remain unknown, post-transcriptional modifications are known to influence RNA stability, interaction with protein partners, and translation capacity. Here, we report the nucleoside modification profile of Ast obtained through liquid chromatography coupled with mass spectrometry (LC-MS) analysis. The epitranscriptome includes a limited set of modified nucleosides but predominantly ribose methylations. A number of these modifications were mapped to specific positions of the sequence through RNA modification mapping procedures. The presence of modifications on Ast is consistent with the RNA-modifying enzymes interacting with Ast The identification and mapping of Ast post-transcriptional modifications is expected to elucidate the mechanisms through which this versatile molecule can carry out diverse activities in different cell compartments. Manipulation of post-transcriptional modifications on the Ast RNA may have therapeutic implications.
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VIH-1 , Cromatografía Liquida , VIH-1/genética , VIH-1/metabolismo , Humanos , Nucleósidos/metabolismo , Procesamiento Postranscripcional del ARN , ARN sin Sentido/genética , Secuencias Repetidas TerminalesRESUMEN
BACKGROUND & AIMS: Despite evidence for the benefits of palliative care (PC) referrals and early advance care planning (ACP) discussions for patients with chronic diseases, patients with end-stage liver disease (ESLD) often do not receive such care. We sought to examine physicians' perceptions of the barriers to PC and timely ACP discussions for patients with ESLD. METHODS: We conducted a cross-sectional survey of hepatologists and gastroenterologists who provide care to adult patients with ESLD, recruited from the American Association for the Study of Liver Diseases 2018 membership registry. Using a questionnaire adapted from prior studies, we assessed physicians' perceptions of barriers to PC use and timely ACP discussions; 396 of 1236 eligible physicians (32%) completed the questionnaire. RESULTS: The most commonly cited barriers to PC use were cultural factors that affect perception of PC (by 95% of respondents), unrealistic expectations from patients about their prognosis (by 93% of respondents), and competing demands for clinicians' time (by 91% of respondents). Most respondents (81%) thought that ACP discussions with patients who have ESLD typically occur too late in the course of illness. The most commonly cited barriers to timely ACP discussions were insufficient communication between clinicians and families about goals of care (by 84% of respondents) and insufficient cultural competency training about end-of-life care (81%). CONCLUSION: There are substantial barriers to use of PC and timely discussions about ACP-most hepatologists and gastroenterologists believe that ACP occurs too late for patients with ESLD. Strategies are needed to overcome barriers and increase delivery of high-quality palliative and end-of-life care to patients with ESLD.
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Planificación Anticipada de Atención , Enfermedad Hepática en Estado Terminal , Gastroenterólogos , Cuidados Paliativos , Relaciones Médico-Paciente , Actitud del Personal de Salud , Comunicación , Estudios Transversales , Competencia Cultural , Femenino , Humanos , Masculino , Encuestas y Cuestionarios , Estados UnidosRESUMEN
N(6)-Methyladenosine (m(6)A) is a widespread, reversible chemical modification of RNA molecules, implicated in many aspects of RNA metabolism. Little quantitative information exists as to either how many transcript copies of particular genes are m(6)A modified ('m(6)A levels') or the relationship of m(6)A modification(s) to alternative RNA isoforms. To deconvolute the m(6)A epitranscriptome, we developed m(6)A-level and isoform-characterization sequencing (m(6)A-LAIC-seq). We found that cells exhibit a broad range of nonstoichiometric m(6)A levels with cell-type specificity. At the level of isoform characterization, we discovered widespread differences in the use of tandem alternative polyadenylation (APA) sites by methylated and nonmethylated transcript isoforms of individual genes. Strikingly, there is a strong bias for methylated transcripts to be coupled with proximal APA sites, resulting in shortened 3' untranslated regions, while nonmethylated transcript isoforms tend to use distal APA sites. m(6)A-LAIC-seq yields a new perspective on transcriptome complexity and links APA usage to m(6)A modifications.
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Adenosina/análogos & derivados , Epigénesis Genética/genética , Epigenómica/métodos , Regulación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Transcriptoma , Regiones no Traducidas 3'/genética , Adenosina/química , Adenosina/genética , Células Cultivadas , Humanos , Poliadenilación , Isoformas de Proteínas , ARN Mensajero/genética , Células Madre/metabolismoRESUMEN
Specialty palliative care (PC) is underused for patients with end-stage liver disease (ESLD). We sought to examine attitudes of hepatologists and gastroenterologists about PC for patients with ESLD. We conducted a cross-sectional survey of these specialists who provide care to patients with ESLD. Participants were recruited from the American Association for the Study of Liver Diseases membership directory. Using a questionnaire adapted from prior studies, we examined physicians' attitudes about PC and whether these attitudes varied based on patients' candidacy for liver transplantation. We identified predictors of physicians' attitudes about PC using linear regression. Approximately one-third of eligible physicians (396/1236, 32%) completed the survey. Most (95%) believed that centers providing care to patients with ESLD should have PC services, and 86% trusted PC clinicians to care for their patients. Only a minority reported collaborating frequently with inpatient (32%) or outpatient (11%) PC services. Most believed that when patients hear the term PC, they feel scared (94%) and anxious (87%). Most (83%) believed that patients would think nothing more could be done for their underlying disease if a PC referral was suggested. Physicians who believed that ESLD is a terminal condition (B = 1.09; P = 0.006) reported more positive attitudes about PC. Conversely, physicians with negative perceptions of PC for transplant candidates (B = -0.22; standard error = 0.05; P < 0.001) reported more negative attitudes toward PC. In conclusion, although most hepatologists and gastroenterologists believe that patients with ESLD should have access to PC, they reported rarely collaborating with PC teams and had substantial concerns about patients' perceptions of PC. Interventions are needed to overcome misperceptions of PC and to promote collaboration with PC clinicians for patients with ESLD.
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Actitud , Enfermedad Hepática en Estado Terminal/terapia , Gastroenterólogos/psicología , Trasplante de Hígado , Cuidados Paliativos/psicología , Estudios Transversales , Enfermedad Hepática en Estado Terminal/psicología , Femenino , Gastroenterólogos/estadística & datos numéricos , Humanos , Colaboración Intersectorial , Masculino , Derivación y Consulta/estadística & datos numéricos , Encuestas y Cuestionarios/estadística & datos numéricos , Factores de Tiempo , Estados Unidos , Listas de EsperaRESUMEN
Many long noncoding RNA (lncRNA) species have been identified in mammalian cells, but the genomic origin and regulation of these molecules in individual cell types is poorly understood. We have generated catalogs of lncRNA species expressed in human and murine embryonic stem cells and mapped their genomic origin. A surprisingly large fraction of these transcripts (>60%) originate from divergent transcription at promoters of active protein-coding genes. The divergently transcribed lncRNA/mRNA gene pairs exhibit coordinated changes in transcription when embryonic stem cells are differentiated into endoderm. Our results reveal that transcription of most lncRNA genes is coordinated with transcription of protein-coding genes.
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Células Madre Embrionarias/metabolismo , ARN Mensajero/genética , ARN no Traducido/genética , Transcripción Genética , Animales , Diferenciación Celular , Células Madre Embrionarias/citología , Humanos , RatonesRESUMEN
Common genetic variants in glucokinase regulator (GCKR), which encodes GKRP, a regulator of hepatic glucokinase (GCK), influence multiple metabolic traits in genome-wide association studies (GWASs), making GCKR one of the most pleiotropic GWAS loci in the genome. It is unclear why. Prior work has demonstrated that GCKR influences the hepatic cytosolic NADH/NAD+ ratio, also referred to as reductive stress. Here, we demonstrate that reductive stress is sufficient to activate the transcription factor ChREBP and necessary for its activation by the GKRP-GCK interaction, glucose, and ethanol. We show that hepatic reductive stress induces GCKR GWAS traits such as increased hepatic fat, circulating FGF21, and circulating acylglycerol species, which are also influenced by ChREBP. We define the transcriptional signature of hepatic reductive stress and show its upregulation in fatty liver disease and downregulation after bariatric surgery in humans. These findings highlight how a GCKR-reductive stress-ChREBP axis influences multiple human metabolic traits.
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Estudio de Asociación del Genoma Completo , Glucoquinasa , Humanos , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Glucoquinasa/genética , Glucoquinasa/metabolismo , Glucosa/metabolismo , Hígado/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Obesity is a major cause of metabolic dysfunction-associated steatohepatitis (MASH) and is characterized by inflammation and insulin resistance. Interferon-γ (IFNγ) is a pro-inflammatory cytokine elevated in obesity and modulating macrophage functions. Here, we show that male mice with loss of IFNγ signaling in myeloid cells (Lyz-IFNγR2-/-) are protected from diet-induced insulin resistance despite fatty liver. Obesity-mediated liver inflammation is also attenuated with reduced interleukin (IL)-12, a cytokine primarily released by macrophages, and IL-12 treatment in vivo causes insulin resistance by impairing hepatic insulin signaling. Following MASH diets, Lyz-IFNγR2-/- mice are rescued from developing liver fibrosis, which is associated with reduced fibroblast growth factor (FGF) 21 levels. These results indicate critical roles for IFNγ signaling in macrophages and their release of IL-12 in modulating obesity-mediated insulin resistance and fatty liver progression to MASH. In this work, we identify the IFNγ-IL12 axis in regulating intercellular crosstalk in the liver and as potential therapeutic targets to treat MASH.
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Hígado Graso , Resistencia a la Insulina , Interferón gamma , Interleucina-12 , Hígado , Macrófagos , Ratones Noqueados , Obesidad , Transducción de Señal , Animales , Interferón gamma/metabolismo , Interleucina-12/metabolismo , Masculino , Obesidad/metabolismo , Ratones , Hígado Graso/metabolismo , Hígado Graso/patología , Macrófagos/metabolismo , Hígado/metabolismo , Hígado/patología , Ratones Endogámicos C57BL , Dieta Alta en Grasa/efectos adversos , Receptores de Interferón/metabolismo , Receptores de Interferón/genética , Receptor de Interferón gamma , Cirrosis Hepática/metabolismo , Cirrosis Hepática/patología , Cirrosis Hepática/genéticaRESUMEN
Background & Aims: Fibrosis is the common endpoint for all forms of chronic liver injury, and progression of fibrosis leads to the development of end-stage liver disease. Activation of hepatic stellate cells (HSCs) and their transdifferentiation to myofibroblasts results in the accumulation of extracellular matrix (ECM) proteins that form the fibrotic scar. Long noncoding (lnc) RNAs regulate the activity of HSCs and may provide targets for fibrotic therapies. Methods: We identified lncRNA TILAM as expressed near COL1A1 in human HSCs and performed loss-of-function studies in human HSCs and liver organoids. Transcriptomic analyses of HSCs isolated from mice defined the murine ortholog of TILAM . We then generated Tilam -deficient GFP reporter mice and quantified fibrotic responses to carbon tetrachloride (CCl 4 ) and choline-deficient L-amino acid defined high fat diet (CDA-HFD). Co-precipitation studies, mass spectrometry, and gene expression analyses identified protein partners of TILAM . Results: TILAM is conserved between human and mouse HSCs and regulates expression of ECM proteins, including collagen. Tilam is selectively induced in HSCs during the development of fibrosis in vivo . In both male and female mice, loss of Tilam results in reduced fibrosis in the setting of CCl 4 and CDA-HFD injury models. TILAM interacts with promyelocytic leukemia protein (PML) to stabilize PML protein levels and promote the fibrotic activity of HSCs. Conclusion: TILAM is activated in HSCs and interacts with PML to drive the development of liver fibrosis. Depletion of TILAM may serve as a therapeutic approach to combat the development of end stage liver disease.
RESUMEN
Chronic liver injury causes fibrosis, characterized by the formation of scar tissue resulting from excessive accumulation of extracellular matrix (ECM) proteins. Hepatic stellate cell (HSC) myofibroblasts are the primary cell type responsible for liver fibrosis, yet there are currently no therapies directed at inhibiting the activity of HSC myofibroblasts. To search for potential anti-fibrotic compounds, we performed a high-throughput compound screen in primary human HSC myofibroblasts and identified 19 small molecules that induce HSC inactivation, including the polyether ionophore nanchangmycin (NCMC). NCMC induces lipid re-accumulation while reducing collagen expression, deposition of collagen in the extracellular matrix, cell proliferation, and migration. We find that NCMC increases cytosolic Ca2+ and reduces the phosphorylated protein levels of FYN, PTK2 (FAK), MAPK1/3 (ERK2/1), HSPB1 (HSP27), and STAT5B. Further, depletion of each of these kinases suppress COL1A1 expression. These studies reveal a signaling network triggered by NCMC to inactivate HSC myofibroblasts and reduce expression of proteins that compose the fibrotic scar. Identification of the antifibrotic effects of NCMC and the elucidation of pathways by which NCMC inhibits fibrosis provide new tools and therapeutic targets that could potentially be utilized to combat the development and progression of liver fibrosis.
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Cicatriz , Células Estrelladas Hepáticas , Cicatriz/patología , Colágeno/metabolismo , Éteres , Proteínas de la Matriz Extracelular/metabolismo , Fibrosis , Quinasa 1 de Adhesión Focal/metabolismo , Células Estrelladas Hepáticas/metabolismo , Humanos , Cirrosis Hepática/tratamiento farmacológico , Cirrosis Hepática/metabolismo , Cirrosis Hepática/patología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Compuestos de EspiroRESUMEN
Long non-coding RNAs (lncRNAs) are important biological mediators that regulate numerous cellular processes. New experimental evidence suggests that lncRNAs play essential roles in liver development, normal liver physiology, fibrosis, and malignancy, including hepatocellular carcinoma and cholangiocarcinoma. In this review, we summarise our current understanding of the function of lncRNAs in the liver in both health and disease, as well as discuss approaches that could be used to target these non-coding transcripts for therapeutic purposes.
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Fusion of nascent myoblasts to pre-existing myofibres is critical for skeletal muscle growth and repair. The vast majority of molecules known to regulate myoblast fusion are necessary in this process. Here, we uncover, through high-throughput in vitro assays and in vivo studies in the chicken embryo, that TGFß (SMAD2/3-dependent) signalling acts specifically and uniquely as a molecular brake on muscle fusion. While constitutive activation of the pathway arrests fusion, its inhibition leads to a striking over-fusion phenotype. This dynamic control of TGFß signalling in the embryonic muscle relies on a receptor complementation mechanism, prompted by the merging of myoblasts with myofibres, each carrying one component of the heterodimer receptor complex. The competence of myofibres to fuse is likely restored through endocytic degradation of activated receptors. Altogether, this study shows that muscle fusion relies on TGFß signalling to regulate its pace.
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Fibras Musculares Esqueléticas/efectos de los fármacos , Mioblastos/citología , Mioblastos/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Comunicación Celular/fisiología , Diferenciación Celular/fisiología , Fusión Celular , Pollos , Inmunohistoquímica , Hibridación in Situ , Ratones , Fibras Musculares Esqueléticas/metabolismo , Miofibrillas/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Cooperation between DNA, RNA and protein regulates gene expression and controls differentiation through interactions that connect regions of nucleic acids and protein domains and through the assembly of biomolecular condensates. Here, we report that endoderm differentiation is regulated by the interaction between the long non-coding RNA (lncRNA) DIGIT and the bromodomain and extraterminal domain protein BRD3. BRD3 forms phase-separated condensates of which the formation is promoted by DIGIT, occupies enhancers of endoderm transcription factors and is required for endoderm differentiation. BRD3 binds to histone H3 acetylated at lysine 18 (H3K18ac) in vitro and co-occupies the genome with H3K18ac. DIGIT is also enriched in regions of H3K18ac, and the depletion of DIGIT results in decreased recruitment of BRD3 to these regions. Our findings show that cooperation between DIGIT and BRD3 at regions of H3K18ac regulates the transcription factors that drive endoderm differentiation and suggest that protein-lncRNA phase-separated condensates have a broader role as regulators of transcription.
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Diferenciación Celular , Endodermo/citología , Histonas/metabolismo , Células Madre Embrionarias Humanas/citología , Transición de Fase , ARN Largo no Codificante/genética , Factores de Transcripción/metabolismo , Acetilación , Endodermo/metabolismo , Genoma Humano , Histonas/genética , Células Madre Embrionarias Humanas/metabolismo , Humanos , Lisina/genética , Lisina/metabolismo , Dominios Proteicos , Procesamiento Proteico-Postraduccional , Factores de Transcripción/genéticaRESUMEN
Hepatic stellate cells (HSCs) drive hepatic fibrosis. Therapies that inactivate HSCs have clinical potential as antifibrotic agents. We previously identified acid ceramidase (aCDase) as an antifibrotic target. We showed that tricyclic antidepressants (TCAs) reduce hepatic fibrosis by inhibiting aCDase and increasing the bioactive sphingolipid ceramide. We now demonstrate that targeting aCDase inhibits YAP/TAZ activity by potentiating its phosphorylation-mediated proteasomal degradation via the ubiquitin ligase adaptor protein ß-TrCP. In mouse models of fibrosis, pharmacologic inhibition of aCDase or genetic knockout of aCDase in HSCs reduces fibrosis, stromal stiffness, and YAP/TAZ activity. In patients with advanced fibrosis, aCDase expression in HSCs is increased. Consistently, a signature of the genes most down-regulated by ceramide identifies patients with advanced fibrosis who could benefit from aCDase targeting. The findings implicate ceramide as a critical regulator of YAP/TAZ signaling and HSC activation and highlight aCDase as a therapeutic target for the treatment of fibrosis.
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Ceramidasa Ácida , Células Estrelladas Hepáticas , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Fibrosis , Células Estrelladas Hepáticas/metabolismo , Humanos , Ratones , Transducción de SeñalRESUMEN
Soon after the discovery of transforming growth factor-ß (TGF-ß), seminal work in vertebrate and invertebrate models revealed the TGF-ß family to be central regulators of tissue morphogenesis. Members of the TGF-ß family direct some of the earliest cell-fate decisions in animal development, coordinate complex organogenesis, and contribute to tissue homeostasis in the adult. Here, we focus on the role of the TGF-ß family in mammalian stem-cell biology and discuss its wide and varied activities both in the regulation of pluripotency and in cell-fate commitment.
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Células Madre Adultas/metabolismo , Diferenciación Celular , Autorrenovación de las Células , Células Madre Embrionarias/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Células Madre Adultas/citología , Animales , Proteínas Morfogenéticas Óseas/metabolismo , Proteínas Morfogenéticas Óseas/fisiología , Células Madre Embrionarias/citología , Regulación del Desarrollo de la Expresión Génica , Humanos , Ratones , Transducción de SeñalRESUMEN
N6-methyladenosine (m6A) is the most abundant internal modification of mRNAs and is implicated in all aspects of post-transcriptional RNA metabolism. However, little is known about m6A modifications to circular (circ) RNAs. We developed a computational pipeline (AutoCirc) that, together with depletion of ribosomal RNA and m6A immunoprecipitation, defined thousands of m6A circRNAs with cell-type-specific expression. The presence of m6A circRNAs is corroborated by interaction between circRNAs and YTHDF1/YTHDF2, proteins that read m6A sites in mRNAs, and by reduced m6A levels upon depletion of METTL3, the m6A writer. Despite sharing m6A readers and writers, m6A circRNAs are frequently derived from exons that are not methylated in mRNAs, whereas mRNAs that are methylated on the same exons that compose m6A circRNAs exhibit less stability in a process regulated by YTHDF2. These results expand our understanding of the breadth of m6A modifications and uncover regulation of circRNAs through m6A modification.
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Adenosina/análogos & derivados , Genoma Humano , ARN/metabolismo , Adenosina/metabolismo , Secuencia de Bases , Biología Computacional , Elementos Transponibles de ADN/genética , Exones/genética , Exorribonucleasas/metabolismo , Regulación de la Expresión Génica , Semivida , Células HeLa , Células Madre Embrionarias Humanas/metabolismo , Humanos , Metilación , Metiltransferasas/metabolismo , ARN/genética , Estabilidad del ARN , ARN Circular , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismoRESUMEN
Activation of hepatic stellate cells (HSCs) in response to injury is a key step in hepatic fibrosis, and is characterized by trans-differentiation of quiescent HSCs to HSC myofibroblasts, which secrete extracellular matrix proteins responsible for the fibrotic scar. There are currently no therapies to directly inhibit hepatic fibrosis. We developed a small molecule screen to identify compounds that inactivate human HSC myofibroblasts through the quantification of lipid droplets. We screened 1600 compounds and identified 21 small molecules that induce HSC inactivation. Four hits were tricyclic antidepressants (TCAs), and they repressed expression of pro-fibrotic factors Alpha-Actin-2 (ACTA2) and Alpha-1 Type I Collagen (COL1A1) in HSCs. RNA sequencing implicated the sphingolipid pathway as a target of the TCAs. Indeed, TCA treatment of HSCs promoted accumulation of ceramide through inhibition of acid ceramidase (aCDase). Depletion of aCDase also promoted accumulation of ceramide and was associated with reduced COL1A1 expression. Treatment with B13, an inhibitor of aCDase, reproduced the antifibrotic phenotype as did the addition of exogenous ceramide. Our results show that detection of lipid droplets provides a robust readout to screen for regulators of hepatic fibrosis and have identified a novel antifibrotic role for ceramide.