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1.
Aging Biol ; 1(1)2023.
Artigo em Inglês | MEDLINE | ID: mdl-38124711

RESUMO

Age is the greatest risk factor for the development of type 2 diabetes mellitus (T2DM). Age-related decline in organ function is attributed to the accumulation of stochastic damage, including damage to the nuclear genome. Islets of T2DM patients display increased levels of DNA damage. However, whether this is a cause or consequence of the disease has not been elucidated. Here, we asked if spontaneous, endogenous DNA damage in ß-cells can drive ß-cell dysfunction and diabetes, via deletion of Ercc1, a key DNA repair gene, in ß-cells. Mice harboring Ercc1-deficient ß-cells developed adult-onset diabetes as demonstrated by increased random and fasted blood glucose levels, impaired glucose tolerance, and reduced insulin secretion. The inability to repair endogenous DNA damage led to an increase in oxidative DNA damage and apoptosis in ß-cells and a significant loss of ß-cell mass. Using electron microscopy, we identified ß-cells in clear distress that showed an increased cell size, enlarged nuclear size, reduced number of mature insulin granules, and decreased number of mitochondria. Some ß-cells were more affected than others consistent with the stochastic nature of spontaneous DNA damage. Ercc1-deficiency in ß-cells also resulted in loss of ß-cell function as glucose-stimulated insulin secretion and mitochondrial function were impaired in islets isolated from mice harboring Ercc1-deficient ß-cells. These data reveal that unrepaired endogenous DNA damage is sufficient to drive ß-cell dysfunction and provide a mechanism by which age increases the risk of T2DM.

3.
Gastroenterology ; 165(1): 228-243.e2, 2023 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-37059338

RESUMO

BACKGROUND & AIMS: We reported that cholangiocyte senescence, regulated by the transcription factor ETS proto-oncogene 1 (ETS1), is a pathogenic feature of primary sclerosing cholangitis (PSC). Furthermore, histone 3 lysine 27 is acetylated at senescence-associated loci. The epigenetic readers, bromodomain and extra-terminal domain (BET) proteins, bind acetylated histones, recruit transcription factors, and drive gene expression. Thus, we tested the hypothesis that BET proteins interact with ETS1 to drive gene expression and cholangiocyte senescence. METHODS: We performed immunofluorescence for BET proteins (BRD2 and 4) in liver tissue from liver tissue from PSC patients and a mouse PSC model. Using normal human cholangiocytes (NHCs), NHCs experimentally induced to senescence (NHCsen), and PSC patient-derived cholangiocytes (PSCDCs), we assessed senescence, fibroinflammatory secretome, and apoptosis after BET inhibition or RNA interference depletion. We assessed BET interaction with ETS1 in NHCsen and tissues from PSC patient, and the effects of BET inhibitors on liver fibrosis, senescence, and inflammatory gene expression in mouse models. RESULTS: Tissue from patients with PSC and a mouse PSC model exhibited increased cholangiocyte BRD2 and 4 protein (∼5×) compared with controls without disease. NHCsen exhibited increased BRD2 and 4 (∼2×), whereas PSCDCs exhibited increased BRD2 protein (∼2×) relative to NHC. BET inhibition in NHCsen and PSCDCs reduced senescence markers and inhibited the fibroinflammatory secretome. ETS1 interacted with BRD2 in NHCsen, and BRD2 depletion diminished NHCsen p21 expression. BET inhibitors reduced senescence, fibroinflammatory gene expression, and fibrosis in the 3,5-diethoxycarbonyl-1,4-dihydrocollidine-fed and Mdr2-/- mouse models. CONCLUSION: Our data suggest that BRD2 is an essential mediator of the senescent cholangiocyte phenotype and is a potential therapeutic target for patients with PSC.


Assuntos
Colangite Esclerosante , Animais , Camundongos , Humanos , Colangite Esclerosante/patologia , Fígado/patologia , Regulação da Expressão Gênica , Histonas/metabolismo , Proto-Oncogenes , Epigênese Genética
5.
Semin Immunopathol ; 44(4): 527-544, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35178659

RESUMO

The cholangiopathies are a group of liver diseases that affect cholangiocytes, the epithelial cells that line the bile ducts. Biliary atresia (BA), primary biliary cholangitis (PBC), and primary sclerosing cholangitis (PSC) are three cholangiopathies with significant immune-mediated pathogenesis where chronic inflammation and fibrosis lead to obliteration of bile ducts and eventual liver cirrhosis. Cellular senescence is a state of cell cycle arrest in which cells become resistant to apoptosis and profusely secrete a bioactive secretome. Recent evidence indicates that cholangiocyte senescence contributes to the pathogenesis of BA, PBC, and PSC. This review explores the role of cholangiocyte senescence in BA, PBC, and PSC, ascertains how cholangiocyte senescence may promote a senescence-associated immunopathology in these cholangiopathies, and provides the rationale for therapeutically targeting senescence as a treatment option for BA, PBC, and PSC.


Assuntos
Colangite Esclerosante , Ductos Biliares/metabolismo , Ductos Biliares/patologia , Senescência Celular , Colangite Esclerosante/etiologia , Colangite Esclerosante/terapia , Células Epiteliais , Fibrose , Humanos
6.
Hepatology ; 75(5): 1110-1122, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-34942041

RESUMO

BACKGROUNDS AND AIMS: Polycystic liver disease (PLD) is characterized by defective cholangiocyte cilia that regulate progressive growth of hepatic cysts. Because formation of primary cilia is influenced by autophagy through degradation of proteins involved in ciliogenesis, we hypothesized that ciliary defects in PLD cholangiocytes (PLDCs) originate from autophagy-mediated depletion of ciliogenic proteins ADP-ribosylation factor-like protein 3 (ARL3) and ADP-ribosylation factor-like protein 13B (ARL13B) and ARL-dependent mislocation of a ciliary-localized bile acid receptor, Takeda G-protein-coupled receptor 5 (TGR5), the activation of which enhances hepatic cystogenesis (HCG). The aims here were to determine whether: (1) ciliogenesis is impaired in PLDC, is associated with increased autophagy, and involves autophagy-mediated depletion of ARL3 and ARL13B; (2) depletion of ARL3 and ARL13B in PLDC cilia impacts ciliary localization of TGR5; and (3) pharmacological inhibition of autophagy re-establishes cholangiocyte cilia and ciliary localization of ARL3, ARL3B, and TGR5 and reduces HCG. APPROACH AND RESULTS: By using liver tissue from healthy persons and patients with PLD, in vitro and in vivo models of PLD, and in vitro models of ciliogenesis, we demonstrated that, in PLDCs: ciliogenesis is impaired; autophagy is enhanced; ARL3 and ARL13B are ubiquitinated by HDAC6, depleted in cilia, and present in autophagosomes; depletion of ARL3 and ARL13B impacts ciliary localization of TGR5; and pharmacological inhibition of autophagy with mefloquine and verteporfin re-establishes cholangiocyte cilia and ciliary localization of ARL3, ARL13B, and TGR5 and reduces HCG. CONCLUSIONS: The intersection between autophagy, defective cholangiocyte cilia, and enhanced HCG contributes to PLD progression and can be considered a target for therapeutic interventions.


Assuntos
Cistos , Fatores de Ribosilação do ADP/metabolismo , Fatores de Ribosilação do ADP/uso terapêutico , Autofagia , Cistos/tratamento farmacológico , Humanos , Fígado/metabolismo , Hepatopatias
7.
Nature ; 594(7861): 100-105, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33981041

RESUMO

Ageing of the immune system, or immunosenescence, contributes to the morbidity and mortality of the elderly1,2. To define the contribution of immune system ageing to organism ageing, here we selectively deleted Ercc1, which encodes a crucial DNA repair protein3,4, in mouse haematopoietic cells to increase the burden of endogenous DNA damage and thereby senescence5-7 in the immune system only. We show that Vav-iCre+/-;Ercc1-/fl mice were healthy into adulthood, then displayed premature onset of immunosenescence characterized by attrition and senescence of specific immune cell populations and impaired immune function, similar to changes that occur during ageing in wild-type mice8-10. Notably, non-lymphoid organs also showed increased senescence and damage, which suggests that senescent, aged immune cells can promote systemic ageing. The transplantation of splenocytes from Vav-iCre+/-;Ercc1-/fl or aged wild-type mice into young mice induced senescence in trans, whereas the transplantation of young immune cells attenuated senescence. The treatment of Vav-iCre+/-;Ercc1-/fl mice with rapamycin reduced markers of senescence in immune cells and improved immune function11,12. These data demonstrate that an aged, senescent immune system has a causal role in driving systemic ageing and therefore represents a key therapeutic target to extend healthy ageing.


Assuntos
Envelhecimento/imunologia , Envelhecimento/fisiologia , Sistema Imunitário/imunologia , Sistema Imunitário/fisiologia , Imunossenescência/imunologia , Imunossenescência/fisiologia , Especificidade de Órgãos/imunologia , Especificidade de Órgãos/fisiologia , Envelhecimento/efeitos dos fármacos , Envelhecimento/patologia , Animais , Dano ao DNA/imunologia , Dano ao DNA/fisiologia , Reparo do DNA/imunologia , Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Feminino , Envelhecimento Saudável/imunologia , Envelhecimento Saudável/fisiologia , Homeostase/imunologia , Homeostase/fisiologia , Sistema Imunitário/efeitos dos fármacos , Imunossenescência/efeitos dos fármacos , Masculino , Camundongos , Especificidade de Órgãos/efeitos dos fármacos , Rejuvenescimento , Sirolimo/farmacologia , Baço/citologia , Baço/transplante
8.
Liver Int ; 40(10): 2455-2468, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32558183

RESUMO

BACKGROUND & AIMS: Primary sclerosing cholangitis (PSC) is a chronic liver disease characterized by peribiliary inflammation and fibrosis. Cholangiocyte senescence is a prominent feature of PSC. Here, we hypothesize that extracellular vesicles (EVs) from senescent cholangiocytes influence the phenotype of target cells. METHODS: EVs were isolated from normal human cholangiocytes (NHCs), cholangiocytes from PSC patients and NHCs experimentally induced to senescence. NHCs, malignant human cholangiocytes (MHCs) and monocytes were exposed to 108 EVs from each donor cell population and assessed for proliferation, MAPK activation and migration. Additionally, we isolated EVs from plasma of wild-type and Mdr2-/- mice (a murine model of PSC), and assessed mouse monocyte activation. RESULTS: EVs exhibited the size and protein markers of exosomes. The number of EVs released from senescent human cholangiocytes was increased; similarly, the EVs in plasma from Mdr2-/- mice were increased. Additionally, EVs from senescent cholangiocytes were enriched in multiple growth factors, including EGF. NHCs exposed to EVs from senescent cholangiocytes showed increased NRAS and ERK1/2 activation. Moreover, EVs from senescent cholangiocytes promoted proliferation of NHCs and MHCs, findings that were blocked by erlotinib, an EGF receptor inhibitor. Furthermore, EVs from senescent cholangiocytes induced EGF-dependent Interleukin 1-beta and Tumour necrosis factor expression and migration of human monocytes; similarly, Mdr2-/- mouse plasma EVs induced activation of mouse monocytes. CONCLUSIONS: The data continue to support the importance of cholangiocyte senescence in PSC pathogenesis, directly implicate EVs in cholangiocyte proliferation, malignant progression and immune cell activation and migration, and identify novel therapeutic approaches for PSC.


Assuntos
Colangite Esclerosante , Vesículas Extracelulares , Animais , Senescência Celular , Receptores ErbB , Humanos , Camundongos , Fenótipo
9.
Am J Pathol ; 190(6): 1284-1297, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32240619

RESUMO

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL; TNFSF10) receptor (TR) is a pro-apoptotic receptor whose contribution to chronic cholestatic liver disease is unclear. Herein, we examined TRAIL receptor signaling in a mouse model of cholestatic liver injury. TRAIL receptor-deficient (Tnsf10 or Tr-/-) mice were crossbred with ATP binding cassette subfamily B member 4-deficient (Abcb4-/-, alias Mdr2-/-) mice. Male and female wild-type, Tr-/-, Mdr2-/-, and Tr-/-Mdr2-/- mice were assessed for liver injury, fibrosis, and ductular reactive (DR) cells. Macrophage subsets were examined by high-dimensional mass cytometry (time-of-flight mass cytometry). Mdr2-/- and Tr-/-Mdr2-/- mice had elevated liver weights and serum alanine transferase values. However, fibrosis was primarily periductular in Mdr2-/- mice, compared with extensive bridging fibrosis in Tr-/-Mdr2-/- mice. DR cell population was greatly expanded in the Tr-/-Mdr2-/- versus Mdr2-/- mice. The expanded DR cell population in Tr-/-Mdr2-/- mice was due to decreased cell loss by apoptosis and not enhanced proliferation. As assessed by time-of-flight mass cytometry, total macrophages were more abundant in Tr-/-Mdr2-/- versus Mdr2-/- mice, suggesting the DR cell population promotes macrophage-associated hepatic inflammation. Inhibition of monocyte-derived recruited macrophages using the CCR2/CCR5 antagonist cenicriviroc in the Mdr2-/- mice resulted in further expansion of the DR cell population. In conclusion, genetic deletion of TRAIL receptor increased the DR cell population, macrophage accumulation, and hepatic fibrosis in the Mdr2-/- model of cholestasis.


Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Cirrose Hepática/genética , Fígado/metabolismo , Macrófagos/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Animais , Colestase/genética , Colestase/metabolismo , Colestase/patologia , Modelos Animais de Doenças , Feminino , Fígado/patologia , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Masculino , Camundongos , Camundongos Knockout , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Transdução de Sinais/fisiologia , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATP
10.
Hepatology ; 71(2): 741-748, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31833071

RESUMO

Cholangiocytes are the target of a group of chronic liver diseases termed the "cholangiopathies," in which cholangiocytes react to exogenous and endogenous insults, leading to disease initiation and progression. In primary sclerosing cholangitis (PSC), the focus of this review, the cholangiocyte response to genetic or environmental insults can lead to a heterogeneous response; that is, a subpopulation acquires a ductular reactive and proliferative phenotype, while another subpopulation undergoes senescence and growth arrest. Both ductular reactive cholangiocytes and senescent cholangiocytes can modify the periductal microenvironment through their ability to secrete various cytokines, chemokines, and growth factors, initiating and perpetuating inflammatory and profibrotic responses. This review discusses the similarities and differences, the interrelationships, and the potential pathogenic roles of these reactive proliferative and senescent cholangiocyte subpopulations in PSC.


Assuntos
Ductos Biliares/citologia , Colangite Esclerosante/patologia , Células Epiteliais/fisiologia , Proliferação de Células , Humanos
11.
J Biol Chem ; 294(49): 18698-18713, 2019 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-31659122

RESUMO

Primary sclerosing cholangitis (PSC) is an idiopathic, progressive cholangiopathy. Cholangiocyte senescence is important in PSC pathogenesis, and we have previously reported that senescence is regulated by the transcription factor ETS proto-oncogene 1 (ETS1) and associated with overexpression of BCL2 like 1 (BCL2L1 or BCL-xL), an anti-apoptotic BCL2-family member. Here, we further explored the mechanisms regulating BCL-xL-mediated, apoptosis resistance in senescent cholangiocytes and uncovered that ETS1 and the histone acetyltransferase E1A-binding protein P300 (EP300 or p300) both promote BCL-xL transcription. Using immunofluorescence, we found that BCL-xL protein expression is increased both in cholangiocytes of livers from individuals with PSC and a mouse model of PSC. Using an in vitro model of lipopolysaccharide-induced senescence in normal human cholangiocytes (NHCs), we found increased BCL-xL mRNA and protein levels, and ChIP-PCRs indicated increased occupancy of ETS1, p300, and histone 3 Lys-27 acetylation (H3K27Ac) at the BCL-xL promoter. Using co-immunoprecipitation and proximity ligation assays, we further demonstrate that ETS1 and p300 physically interact in senescent but not control NHCs. Additionally, mutagenesis of predicted ETS1-binding sites within the BCL-xL promoter blocked luciferase reporter activity, and CRISPR/Cas9-mediated genetic deletion of ETS1 reduced senescence-associated BCL-xL expression. In senescent NHCs, TRAIL-mediated apoptosis was reduced ∼70%, and ETS1 deletion or RNAi-mediated BCL-xL suppression increased apoptosis. Overall, our results suggest that ETS1 and p300 promote senescent cholangiocyte resistance to apoptosis by modifying chromatin and inducing BCL-xL expression. These findings reveal ETS1 as a central regulator of both cholangiocyte senescence and the associated apoptosis-resistant phenotype.


Assuntos
Apoptose/efeitos dos fármacos , Proteína Proto-Oncogênica c-ets-1/metabolismo , Fatores de Transcrição/metabolismo , Proteína bcl-X/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Animais , Apoptose/genética , Senescência Celular/efeitos dos fármacos , Senescência Celular/genética , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Lipopolissacarídeos/farmacologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Camundongos , Proto-Oncogene Mas , Proteína Proto-Oncogênica c-ets-1/genética , Fatores de Transcrição/genética , Proteína bcl-X/metabolismo , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATP
12.
Redox Biol ; 17: 259-273, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29747066

RESUMO

Accumulation of senescent cells over time contributes to aging and age-related diseases. However, what drives senescence in vivo is not clear. Here we used a genetic approach to determine if spontaneous nuclear DNA damage is sufficient to initiate senescence in mammals. Ercc1-/∆ mice with reduced expression of ERCC1-XPF endonuclease have impaired capacity to repair the nuclear genome. Ercc1-/∆ mice accumulated spontaneous, oxidative DNA damage more rapidly than wild-type (WT) mice. As a consequence, senescent cells accumulated more rapidly in Ercc1-/∆ mice compared to repair-competent animals. However, the levels of DNA damage and senescent cells in Ercc1-/∆ mice never exceeded that observed in old WT mice. Surprisingly, levels of reactive oxygen species (ROS) were increased in tissues of Ercc1-/∆ mice to an extent identical to naturally-aged WT mice. Increased enzymatic production of ROS and decreased antioxidants contributed to the elevation in oxidative stress in both Ercc1-/∆ and aged WT mice. Chronic treatment of Ercc1-/∆ mice with the mitochondrial-targeted radical scavenger XJB-5-131 attenuated oxidative DNA damage, senescence and age-related pathology. Our findings indicate that nuclear genotoxic stress arises, at least in part, due to mitochondrial-derived ROS, and this spontaneous DNA damage is sufficient to drive increased levels of ROS, cellular senescence, and the consequent age-related physiological decline.


Assuntos
Envelhecimento/genética , Senescência Celular/genética , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Mitocôndrias/genética , Animais , Antioxidantes/metabolismo , Senescência Celular/fisiologia , Óxidos N-Cíclicos/farmacologia , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Humanos , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/genética , Espécies Reativas de Oxigênio/metabolismo
13.
J Hepatol ; 69(3): 676-686, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29802947

RESUMO

BACKGROUND & AIMS: Macrophages contribute to liver disease, but their role in cholestatic liver injury, including primary sclerosing cholangitis (PSC), is unclear. We tested the hypothesis that macrophages contribute to the pathogenesis of, and are therapeutic targets for, PSC. METHODS: Immune cell profile, hepatic macrophage number, localization and polarization, fibrosis, and serum markers of liver injury and cholestasis were measured in an acute (intrabiliary injection of the inhibitor of apoptosis antagonist BV6) and chronic (Mdr2-/- mice) mouse model of sclerosing cholangitis (SC). Selected observations were confirmed in liver specimens from patients with PSC. Because of the known role of the CCR2/CCL2 axis in monocyte/macrophage chemotaxis, therapeutic effects of the CCR2/5 antagonist cenicriviroc (CVC), or genetic deletion of CCR2 (Ccr2-/- mice) were determined in BV6-injected mice. RESULTS: We found increased peribiliary pro-inflammatory (M1-like) and alternatively-activated (M2-like) monocyte-derived macrophages in PSC compared to normal livers. In both SC models, genetic profiling of liver immune cells identified a predominance of monocytes/macrophages; immunohistochemistry confirmed peribiliary monocyte-derived macrophage recruitment (M1>M2-polarized), which paralleled injury onset and was reversed upon resolution in acute SC mice. PSC, senescent and BV6-treated human cholangiocytes released monocyte chemoattractants (CCL2, IL-8) and macrophage-activating factors in vitro. Pharmacological inhibition of monocyte recruitment by CVC treatment or CCR2 genetic deletion attenuated macrophage accumulation, liver injury and fibrosis in acute SC. CONCLUSIONS: Peribiliary recruited macrophages are a feature of both PSC and acute and chronic murine SC models. Pharmacologic and genetic inhibition of peribiliary macrophage recruitment decreases liver injury and fibrosis in mouse SC. These observations suggest monocyte-derived macrophages contribute to the development of SC in mice and in PSC pathogenesis, and support their potential as a therapeutic target. LAY SUMMARY: Primary sclerosing cholangitis (PSC) is an inflammatory liver disease which often progresses to liver failure. The cause of the disease is unclear and therapeutic options are limited. Therefore, we explored the role of white blood cells termed macrophages in PSC given their frequent contribution to other human inflammatory diseases. Our results implicate macrophages in PSC and PSC-like diseases in mice. More importantly, we found that pharmacologic inhibition of macrophage recruitment to the liver reduces PSC-like liver injury in the mouse. These exciting observations highlight potential new strategies to treat PSC.


Assuntos
Quimiocina CCL2/metabolismo , Colangite Esclerosante , Imidazóis/farmacologia , Cirrose Hepática , Macrófagos , Receptores CCR2/metabolismo , Receptores CCR5/metabolismo , Animais , Antagonistas dos Receptores CCR5/farmacologia , Quimiotaxia/efeitos dos fármacos , Quimiotaxia/imunologia , Colangite Esclerosante/tratamento farmacológico , Colangite Esclerosante/imunologia , Colangite Esclerosante/patologia , Modelos Animais de Doenças , Fígado/imunologia , Fígado/patologia , Cirrose Hepática/imunologia , Cirrose Hepática/patologia , Cirrose Hepática/prevenção & controle , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Sulfóxidos , Resultado do Tratamento
14.
Hepatology ; 68(2): 561-573, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29406621

RESUMO

Cholangiocytes normally express primary cilia, a multisensory organelle that detects signals from the cellular environment. Cilia are significantly reduced in cholangiocarcinoma (CCA) by a mechanism involving overexpression of histone deacetylase 6 (HDAC6). Despite HDAC6 overexpression in CCA, we found no differences in its mRNA level, suggesting a posttranscriptional regulation, possibly involving microRNAs (miRNAs). Here, we describe that at least two HDAC6-targeting miRNAs, miR-433 and miR-22, are down-regulated in CCA both in vitro and in vivo. Experimental restoration of these miRNAs in CCA cells reduced HDAC6 expression, induced ciliary restoration, and decreased the malignant phenotype. Furthermore, in contrast to the mature forms, levels of precursor forms of these miRNAs were higher in CCA compared to normal cholangiocytes and accumulated in the nuclei, suggesting a defective nuclear export. We assessed the expression of Exportin-5, the protein responsible for transporting miRNA precursors out of the nucleus, and found it to be reduced by 50% in CCA compared to normal cholangiocytes. Experimental overexpression of Exportin-5 in CCA cells restored precursor and mature forms of these miRNAs to normal levels, inducing a decrease in the expression of HDAC6 and a decrease in the malignant phenotype. Conversely, short hairpin RNA (shRNA) depletion of Exportin-5 in normal cholangiocytes resulted in increased nuclear retention of precursor miRNAs, decreased mature miRNAs, increased cell proliferation, and shorter cilia. CONCLUSION: These data suggest that down-regulated Exportin-5 impairs the nuclear export of miR-433 and miR-22 precursor forms, causing a decrease in levels of mature miR-433 and miR-22 forms, and leading to overexpression of HDAC6 and ciliary loss in CCA. (Hepatology 2018).


Assuntos
Neoplasias dos Ductos Biliares/metabolismo , Colangiocarcinoma/metabolismo , Desacetilase 6 de Histona/metabolismo , MicroRNAs/metabolismo , Neoplasias dos Ductos Biliares/patologia , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células , Colangiocarcinoma/patologia , Cílios , Imunofluorescência , Regulação Neoplásica da Expressão Gênica , Humanos , Hibridização in Situ Fluorescente , Carioferinas/metabolismo , Reação em Cadeia da Polimerase em Tempo Real
15.
Hepatology ; 67(1): 247-259, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28802066

RESUMO

Cholangiocyte senescence has been linked to primary sclerosing cholangitis (PSC). Persistent secretion of growth factors by senescent cholangiocytes leads to the activation of stromal fibroblasts (ASFs), which are drivers of fibrosis. The activated phenotype of ASFs is characterized by an increased sensitivity to apoptotic stimuli. Here, we examined the mechanisms of apoptotic priming in ASFs and explored a combined targeting strategy to deplete senescent cholangiocytes and ASFs from fibrotic tissue to ameliorate liver fibrosis. Using a coculture system, we determined that senescent cholangiocytes promoted quiescent mesenchymal cell activation in a platelet-derived growth factor (PDGF)-dependent manner. We also identified B-cell lymphoma-extra large (Bcl-xL) as a key survival factor in PDGF-activated human and mouse fibroblasts. Bcl-xL was also up-regulated in senescent cholangiocytes. In vitro, inhibition of Bcl-xL by the small molecule Bcl-2 homology domain 3 mimetic, A-1331852, or Bcl-xL-specific small interfering RNA induced apoptosis in PDGF-activated fibroblasts, but not in quiescent fibroblasts. Likewise, inhibition of Bcl-xL reduced the survival and increased apoptosis of senescent cholangiocytes, compared to nonsenescent cells. Treatment of multidrug resistance 2 gene knockout (Mdr2-/- ) mice with A-1331852 resulted in an 80% decrease in senescent cholangiocytes, a reduction of fibrosis-inducing growth factors and cytokines, decrease of α-smooth muscle actin-positive ASFs, and finally in a significant reduction of liver fibrosis. CONCLUSION: Bcl-xL is a key survival factor in ASFs as well as in senescent cholangiocytes. Treatment with the Bcl-xL-specific inhibitor, A-1331852, reduces liver fibrosis, possibly by a dual effect on activated fibroblasts and senescent cholangiocytes. This mechanism represents an attractive therapeutic strategy in biliary fibrosis. (Hepatology 2018;67:247-259).


Assuntos
Benzotiazóis/farmacologia , Ductos Biliares/citologia , Colangite Esclerosante/patologia , Fibroblastos/efeitos dos fármacos , Isoquinolinas/farmacologia , Fator de Crescimento Derivado de Plaquetas/efeitos dos fármacos , Animais , Biópsia por Agulha , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Colangite Esclerosante/tratamento farmacológico , Modelos Animais de Doenças , Resistência a Múltiplos Medicamentos , Fibroblastos/metabolismo , Fibroblastos/patologia , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Terapia de Alvo Molecular , Fator de Crescimento Derivado de Plaquetas/metabolismo , Distribuição Aleatória , Valores de Referência
16.
J Biol Chem ; 292(12): 4833-4846, 2017 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-28184004

RESUMO

Primary sclerosing cholangitis (PSC) is a chronic, fibroinflammatory cholangiopathy (disease of the bile ducts) of unknown pathogenesis. We reported that cholangiocyte senescence features prominently in PSC and that neuroblastoma RAS viral oncogene homolog (NRAS) is activated in PSC cholangiocytes. Additionally, persistent microbial insult (e.g. LPSs) induces cyclin-dependent kinase inhibitor 2A (CDKN2A/p16INK4a) expression and senescence in cultured cholangiocytes in an NRAS-dependent manner. However, the molecular mechanisms involved in LPS-induced cholangiocyte senescence and NRAS-dependent regulation of CDKN2A remain unclear. Using our in vitro senescence model, we found that LPS-induced CDKN2A expression coincided with a 4.5-fold increase in ETS1 (ETS proto-oncogene 1) mRNA, suggesting that ETS1 is involved in regulating CDKN2A This idea was confirmed by RNAi-mediated suppression or genetic deletion of ETS1, which blocked CDKN2A expression and reduced cholangiocyte senescence. Furthermore, site-directed mutagenesis of a predicted ETS-binding site within the CDKN2A promoter abolished luciferase reporter activity. Pharmacological inhibition of RAS/MAPK reduced ETS1 and CDKN2A protein expression and CDKN2A promoter-driven luciferase activity by ∼50%. In contrast, constitutively active NRAS expression induced ETS1 and CDKN2A protein expression, whereas ETS1 RNAi blocked this increase. Chromatin immunoprecipitation-PCR detected increased ETS1 and histone 3 lysine 4 trimethylation (H3K4Me3) at the CDKN2A promoter following LPS-induced senescence. Additionally, phospho-ETS1 expression was increased in cholangiocytes of human PSC livers and in the Abcb4 (Mdr2)-/- mouse model of PSC. These data pinpoint ETS1 and H3K4Me3 as key transcriptional regulators in NRAS-induced expression of CDKN2A, and this regulatory axis may therefore represent a potential therapeutic target for PSC treatment.


Assuntos
Colangite Esclerosante/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Proteína Proto-Oncogênica c-ets-1/genética , Ativação Transcricional , Regulação para Cima , Animais , Linhagem Celular , Senescência Celular , Colangite Esclerosante/imunologia , Colangite Esclerosante/patologia , Inibidor p16 de Quinase Dependente de Ciclina/imunologia , Humanos , Lipopolissacarídeos/imunologia , Fígado/citologia , Fígado/metabolismo , Fígado/patologia , Camundongos , Proto-Oncogene Mas , Proteína Proto-Oncogênica c-ets-1/imunologia , RNA Mensageiro/genética
17.
Hepatology ; 63(1): 185-96, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26044703

RESUMO

UNLABELLED: Primary sclerosing cholangitis (PSC) is a chronic, idiopathic, fibroinflammatory cholangiopathy. The role of the microbiota in PSC etiopathogenesis may be fundamentally important, yet remains obscure. We tested the hypothesis that germ-free (GF) mutltidrug resistance 2 knockout (mdr2(-/-) ) mice develop a distinct PSC phenotype, compared to conventionally housed (CV) mdr2(-/-) mice. Mdr2(-/-) mice (n = 12) were rederived as GF by embryo transfer, maintained in isolators, and sacrificed at 60 days in parallel with age-matched CV mdr2(-/-) mice. Serum biochemistries, gallbladder bile acids, and liver sections were examined. Histological findings were validated morphometrically, biochemically, and by immunofluorescence microscopy (IFM). Cholangiocyte senescence was assessed by p16(INK4a) in situ hybridization in liver tissue and by senescence-associated ß-galactosidase staining in a culture-based model of insult-induced senescence. Serum biochemistries, including alkaline phosphatase, aspartate aminotransferase, and bilirubin, were significantly higher in GF mdr2(-/-) (P < 0.01). Primary bile acids were similar, whereas secondary bile acids were absent, in GF mdr2(-/-) mice. Fibrosis, ductular reaction, and ductopenia were significantly more severe histopathologically in GF mdr2(-/-) mice (P < 0.01) and were confirmed by hepatic morphometry, hydroxyproline assay, and IFM. Cholangiocyte senescence was significantly increased in GF mdr2(-/-) mice and abrogated in vitro by ursodeoxycholic acid (UDCA) treatment. CONCLUSIONS: GF mdr2(-/-) mice exhibit exacerbated biochemical and histological features of PSC and increased cholangiocyte senescence, a characteristic and potential mediator of progressive biliary disease. UDCA, a commensal microbial metabolite, abrogates senescence in vitro. These findings demonstrate the importance of the commensal microbiota and its metabolites in protecting against biliary injury and suggest avenues for future studies of biomarkers and therapeutic interventions in PSC.


Assuntos
Colangite Esclerosante/etiologia , Microbioma Gastrointestinal/fisiologia , Animais , Modelos Animais de Doenças , Progressão da Doença , Feminino , Masculino , Camundongos , Camundongos Knockout
18.
PLoS One ; 10(4): e0125793, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25915403

RESUMO

Cholangiocytes (biliary epithelial cells) actively participate in microbe-induced proinflammatory responses in the liver and contribute to inflammatory and infectious cholangiopathies. We previously demonstrated that cholangiocyte TLR-dependent NRas activation contributes to proinflammatory/ proliferative responses. We test the hypothesis that LPS-induced activation of NRas requires the EGFR. SV40-transformed human cholangiocytes (H69 cells), or low passage normal human cholangiocytes (NHC), were treated with LPS in the presence or absence of EGFR or ADAM metallopeptidase domain 17 (TACE) inhibitors. Ras activation assays, quantitative RT-PCR, and proliferation assays were performed in cells cultured with or without inhibitors or an siRNA to Grb2. Immunofluorescence for phospho-EGFR was performed on LPS-treated mouse samples and specimens from patients with primary sclerosing cholangitis, primary biliary cirrhosis, hepatitis C, and normal livers. LPS-treatment induced an association between the TLR/MyD88 and EGFR/Grb2 signaling apparatus, NRas activation, and EGFR phosphorylation. NRas activation was sensitive to EGFR and TACE inhibitors and correlated with EGFR phosphorylation. The TACE inhibitor and Grb2 depletion prevented LPS-induced IL6 expression (p<0.05) and proliferation (p<0.01). Additionally, cholangiocytes from LPS-treated mouse livers and human primary sclerosing cholangitis (PSC) livers exhibited increased phospho-EGFR (p<0.01). Moreover, LPS-induced mouse cholangiocyte proliferation was inhibited by concurrent treatment with the EGFR inhibitor, Erlotinib. Our results suggest that EGFR is essential for LPS-induced, TLR4/MyD88-mediated NRas activation and induction of a robust proinflammatory cholangiocyte response. These findings have implications not only for revealing the signaling potential of TLRs, but also implicate EGFR as an integral component of cholangiocyte TLR-induced proinflammatory processes.


Assuntos
Ductos Biliares/citologia , Células Epiteliais/efeitos dos fármacos , Receptores ErbB/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Lipopolissacarídeos/farmacologia , Proteínas de Membrana/metabolismo , Proteínas ADAM/antagonistas & inibidores , Proteínas ADAM/metabolismo , Proteína ADAM17 , Animais , Ductos Biliares/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Doenças do Sistema Digestório/metabolismo , Doenças do Sistema Digestório/patologia , Inibidores Enzimáticos/farmacologia , Células Epiteliais/metabolismo , GTP Fosfo-Hidrolases/genética , Humanos , Lipopolissacarídeos/efeitos adversos , Fígado/citologia , Fígado/metabolismo , Fígado/patologia , Proteínas de Membrana/genética , Camundongos , Fosforilação , Transdução de Sinais/efeitos dos fármacos
19.
Lab Invest ; 94(10): 1126-33, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25046437

RESUMO

Primary sclerosing cholangitis (PSC) is a chronic, idiopathic cholangiopathy. The role of cholangiocytes (biliary epithelial cells) in PSC pathogenesis is unknown and remains an active area of research. Here, through cellular, molecular and next-generation sequencing (NGS) methods, we characterize and identify phenotypic and signaling features of isolated PSC patient-derived cholangiocytes. We isolated cholangiocytes from stage 4 PSC patient liver explants by dissection, differential filtration and immune-magnetic bead separation. We maintained cholangiocytes in culture and assessed for: (i) cholangiocyte, cell adhesion and inflammatory markers; (ii) proliferation rate; (iii) transepithelial electrical resistance (TEER); (iv) cellular senescence; and (v) transcriptomic profiles by NGS. We used two well-established normal human cholangiocyte cell lines (H69 and NHC) as controls. Isolated PSC cells expressed cholangiocyte (eg, cytokeratin 7 and 19) and epithelial cell adhesion markers (EPCAM, ICAM) and were negative for hepatocyte and myofibroblast markers (albumin, α-actin). Proliferation rate was lower for PSC compared with normal cholangiocytes (4 vs 2 days, respectively, P<0.01). Maximum TEER was also lower in PSC compared with normal cholangiocytes (100 vs 145 Ωcm(2), P<0.05). Interleukin-6 (IL-6) and IL-8 (protein and mRNA) were both increased compared with NHCs and H69s (all P<0.01). The proportion of cholangiocytes staining positive for senescence-associated ß-galactosidase was higher in PSC cholangiocytes compared with NHCs (48% vs 5%, P<0.01). Finally, NGS confirmed cholangiocyte marker expression in isolated PSC cholangiocytes and extended our findings regarding pro-inflammatory and senescence-associated signaling. In conclusion, we have demonstrated that high-purity cholangiocytes can be isolated from human PSC liver and grown in primary culture. Isolated PSC cholangiocytes exhibit a phenotype that may reflect their in vivo contribution to disease and serve as a vital tool for in vitro investigation of biliary pathobiology and identification of new therapeutic targets in PSC.


Assuntos
Colangite Esclerosante/patologia , Fígado/patologia , Biomarcadores/metabolismo , Linhagem Celular Tumoral , Separação Celular , Senescência Celular , Feminino , Humanos , Junções Intercelulares/patologia , Fígado/metabolismo , Masculino , Pessoa de Meia-Idade
20.
Hepatology ; 59(6): 2263-75, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24390753

RESUMO

UNLABELLED: Primary sclerosing cholangitis (PSC) is an incurable cholangiopathy of unknown etiopathogenesis. Here we tested the hypothesis that cholangiocyte senescence is a pathophysiologically important phenotype in PSC. We assessed markers of cellular senescence and senescence-associated secretory phenotype (SASP) in livers of patients with PSC, primary biliary cirrhosis, hepatitis C, and in normals by fluorescent in situ hybridization (FISH) and immunofluorescence microscopy (IFM). We tested whether endogenous and exogenous biliary constituents affect senescence and SASP in cultured human cholangiocytes. We determined in coculture whether senescent cholangiocytes induce senescence in bystander cholangiocytes. Finally, we explored signaling mechanisms involved in cholangiocyte senescence and SASP. In vivo, PSC cholangiocytes expressed significantly more senescence-associated p16(INK4a) and γH2A.x compared to the other three conditions; expression of profibroinflammatory SASP components (i.e., IL-6, IL-8, CCL2, PAI-1) was also highest in PSC cholangiocytes. In vitro, several biologically relevant endogenous (e.g., cholestane 3,5,6 oxysterol) and exogenous (e.g., lipopolysaccharide) molecules normally present in bile induced cholangiocyte senescence and SASP. Furthermore, experimentally induced senescent human cholangiocytes caused senescence in bystander cholangiocytes. N-Ras, a known inducer of senescence, was increased in PSC cholangiocytes and in experimentally induced senescent cultured cholangiocytes; inhibition of Ras abrogated experimentally induced senescence and SASP. CONCLUSION: Cholangiocyte senescence induced by biliary constituents by way of N-Ras activation is an important pathogenic mechanism in PSC. Pharmacologic inhibition of N-Ras with a resultant reduction in cholangiocyte senescence and SASP is a new therapeutic approach for PSC.


Assuntos
Senescência Celular , Colangite Esclerosante/fisiopatologia , Proteínas ras/metabolismo , Adulto , Secreções Corporais , Estudos de Casos e Controles , Células Cultivadas , Colangite Esclerosante/etiologia , Colangite Esclerosante/metabolismo , Ativação Enzimática , Genes ras , Humanos , Pessoa de Meia-Idade , Fenótipo , Proteínas ras/antagonistas & inibidores
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