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BACKGROUND AIMS: The primary cilium, an organelle that protrudes from cell surfaces, is essential for sensing extracellular signals. With disturbed cellular communication and chronic liver pathologies, this organelle's dysfunctions have been linked to disorders, including polycystic liver disease (PLD) and Cholangiocarcinoma (CCA). The goal of this study was to elucidate the relationship between primary cilia and the crucial regulator of cellular proliferation, the epidermal growth factor receptor (EGFR) signaling pathway, which has been associated with various clinical conditions. APPROACH RESULTS: The study identified aberrant EGFR signaling pathways in cholangiocytes lacking functional primary cilia. Using liver-specific IFT88 knockout mice, a Pkhd1 mutant rat model, and human cell lines that didn't have functional cilia. Cilia-deficient cholangiocytes showed persistent EGFR activation because of impaired receptor degradation, in contrast to their normal counterparts, where EGFR localization to the cilia promotes appropriate signaling. Using HDAC6 inhibitors to restore primary cilia accelerates EGFR degradation, thereby reducing maladaptive signaling. Importantly, experimental intervention with the HDAC6 inhibitor tubastatin A in an orthotopic rat model moved EGFR to cilia and reduced ERK phosphorylation. Concurrent administration of EGFR and HDAC6 inhibitors in cholangiocarcinoma and polycystic liver disease cells demonstrated synergistic anti-proliferative effects, which were associated with the restoration of functioning primary cilia. CONCLUSION: This study's findings shed light on ciliary function and robust EGFR signaling with slower receptor turnover. We could use therapies that restore the function of primary cilia to treat EGFR-driven diseases in PLD and CCA.
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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.
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Cistos , Fatores de Ribosilação do ADP/metabolismo , Fatores de Ribosilação do ADP/uso terapêutico , Autofagia , Cistos/tratamento farmacológico , Humanos , Fígado/metabolismo , HepatopatiasRESUMO
Hepatic cystogenesis in polycystic liver disease (PLD) is associated with abnormalities in multiple cellular processes, including elevated cAMP and overexpression of histone deacetylase 6 (HDAC6). Disease progression in polycystic kidney (PCK) rats (an animal model of PLD) is attenuated by inhibition of either cAMP production or HDAC6. Therefore, we hypothesized that concurrent targeting of HDAC6 and cAMP would synergistically reduce cyst growth. Changes in hepatorenal cystogenesis were examined in PCK rats treated with a pan-HDAC inhibitor, panobinostat; three specific HDAC6 inhibitors, ACY-1215, ACY-738, and ACY-241; and a combination of ACY-1215 and the somatostatin receptor analogue, pasireotide. We also assessed effects of ACY-1215 and pasireotide alone and in combination on cell proliferation, cAMP production, and expression of acetylated α-tubulin in vitro in cultured cholangiocytes and the length of primary cilia and the frequency of ciliated cholangiocytes in vivo in PCK rats. Panobinostat and all three HDAC6 inhibitors decreased hepatorenal cystogenesis in PCK rats. ACY-1215 was more effective than other HDAC inhibitors and was chosen for combinational treatment. ACY-1215 + pasireotide combination synergistically reduced cyst growth and increased length of primary cilia in PCK rats. In cultured cystic cholangiocytes, ACY-1215 + pasireotide combination concurrently decreased cell proliferation and inhibited cAMP levels. These data suggest that the combination of drugs that inhibit HDAC6 and cAMP may be an effective therapy for PLD.
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Cistos/tratamento farmacológico , Desacetilase 6 de Histona/antagonistas & inibidores , Inibidores de Histona Desacetilases/uso terapêutico , Hepatopatias/tratamento farmacológico , Receptores de Somatostatina/agonistas , Acetilação , Animais , Ductos Biliares/efeitos dos fármacos , Ductos Biliares/metabolismo , Ductos Biliares/patologia , Proliferação de Células/efeitos dos fármacos , Cílios/metabolismo , AMP Cíclico/metabolismo , Cistos/patologia , Modelos Animais de Doenças , Sinergismo Farmacológico , Feminino , Desacetilase 6 de Histona/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Ácidos Hidroxâmicos/uso terapêutico , Hepatopatias/patologia , Masculino , Panobinostat/farmacologia , Panobinostat/uso terapêutico , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Ratos , Somatostatina/análogos & derivados , Somatostatina/farmacologia , Somatostatina/uso terapêutico , Tubulina (Proteína)/metabolismoRESUMO
Polycystic liver disease (PLD) is a group of genetic disorders with limited treatment options and significant morbidity. Hepatic cysts arise from cholangiocytes exhibiting a hyperproliferative phenotype. Considering that hyperproliferation of many cell types is associated with alterations in autophagy, we hypothesized that autophagy is altered in PLD cholangiocytes, contributes to hepatic cystogenesis, and might represent a potential therapeutic target. We employed functional pathway cluster analysis and next-generation sequencing, transmission electron microscopy, immunofluorescence confocal microscopy, and western blotting to assess autophagy in human and rodent PLD cholangiocytes. A three-dimensional culture model was used to study the effects of molecular and pharmacologic inhibition of autophagy on hepatic cystogenesis in vitro, and the polycystic kidney disease-specific rat, an animal model of PLD, to study the effects of hydroxychloroquine, a drug that interferes with the autophagy pathway, on disease progression in vivo. Assessment of the transcriptome of PLD cholangiocytes followed by functional pathway cluster analysis revealed that the autophagy-lysosomal pathway is one of the most altered pathways in PLD. Direct evaluation of autophagy in PLD cholangiocytes both in vitro and in vivo showed increased number and size of autophagosomes, lysosomes, and autolysosomes; overexpression of autophagy-related proteins (Atg5, Beclin1, Atg7, and LC3); and enhanced autophagic flux associated with activation of the cAMP-protein kinase A-cAMP response element-binding protein signaling pathway. Molecular and pharmacologic intervention in autophagy with ATG7 small interfering RNA, bafilomycin A1 , and hydroxychloroquine reduced proliferation of PLD cholangiocytes in vitro and growth of hepatic cysts in three-dimensional cultures. Hydroxychloroquine also efficiently inhibited hepatic cystogenesis in the polycystic kidney disease-specific rat. CONCLUSION: Autophagy is increased in PLD cholangiocytes, contributes to hepatic cystogenesis, and represents a potential therapeutic target for disease treatment. (Hepatology 2018;67:1088-1108).
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Autofagia/efeitos dos fármacos , Ductos Biliares/citologia , Cistos/fisiopatologia , Hepatopatias/fisiopatologia , Fígado/patologia , Animais , Autofagia/genética , Autofagia/fisiologia , Ductos Biliares/metabolismo , Western Blotting , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Análise por Conglomerados , Cistos/tratamento farmacológico , Cistos/metabolismo , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Células Epiteliais/metabolismo , Feminino , Imunofluorescência , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Hidroxicloroquina/farmacologia , Fígado/metabolismo , Hepatopatias/metabolismo , Masculino , Microscopia Eletrônica de Transmissão , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Somatostatina/análogos & derivados , Somatostatina/farmacologiaRESUMO
Hepatic cystogenesis in polycystic liver disease is associated with increased levels of cyclic adenosine monophosphate (cAMP) in cholangiocytes lining liver cysts. Takeda G protein receptor 5 (TGR5), a G protein-coupled bile acid receptor, is linked to cAMP and expressed in cholangiocytes. Therefore, we hypothesized that TGR5 might contribute to disease progression. We examined expression of TGR5 and Gα proteins in cultured cholangiocytes and in livers of animal models and humans with polycystic liver disease. In vitro, we assessed cholangiocyte proliferation, cAMP levels, and cyst growth in response to (1) TGR5 agonists (taurolithocholic acid, oleanolic acid [OA], and two synthetic compounds), (2) a novel TGR5 antagonist (m-tolyl 5-chloro-2-[ethylsulfonyl] pyrimidine-4-carboxylate [SBI-115]), and (3) a combination of SBI-115 and pasireotide, a somatostatin receptor analogue. In vivo, we examined hepatic cystogenesis in OA-treated polycystic kidney rats and after genetic elimination of TGR5 in double mutant TGR5-/- ;Pkhd1del2/del2 mice. Compared to control, expression of TGR5 and Gαs (but not Gαi and Gαq ) proteins was increased 2-fold to 3-fold in cystic cholangiocytes in vitro and in vivo. In vitro, TGR5 stimulation enhanced cAMP production, cell proliferation, and cyst growth by â¼40%; these effects were abolished after TGR5 reduction by short hairpin RNA. OA increased cystogenesis in polycystic kidney rats by 35%; in contrast, hepatic cystic areas were decreased by 45% in TGR5-deficient TGR5-/- ;Pkhd1del2/del2 mice. TGR5 expression and its colocalization with Gαs were increased â¼2-fold upon OA treatment. Levels of cAMP, cell proliferation, and cyst growth in vitro were decreased by â¼30% in cystic cholangiocytes after treatment with SBI-115 alone and by â¼50% when SBI-115 was combined with pasireotide. CONCLUSION: TGR5 contributes to hepatic cystogenesis by increasing cAMP and enhancing cholangiocyte proliferation; our data suggest that a TGR5 antagonist alone or concurrently with somatostatin receptor agonists represents a potential therapeutic approach in polycystic liver disease. (Hepatology 2017;66:1197-1218).
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AMP Cíclico/metabolismo , Cistos/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Hepatopatias/metabolismo , Pirimidinas/uso terapêutico , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proliferação de Células/efeitos dos fármacos , Cistos/tratamento farmacológico , Avaliação Pré-Clínica de Medicamentos , Quimioterapia Combinada , Humanos , Hepatopatias/tratamento farmacológico , Camundongos , Ácido Oleanólico , Doenças Renais Policísticas/metabolismo , Cultura Primária de Células , Pirimidinas/farmacologia , Ratos , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/genética , Somatostatina/análogos & derivados , Somatostatina/farmacologia , Somatostatina/uso terapêuticoRESUMO
BACKGROUND & AIMS: Polycystic liver diseases (PLDs) are genetic disorders characterized by progressive biliary cystogenesis. Current therapies show short-term and/or modest beneficial effects. Cystic cholangiocytes hyperproliferate as a consequence of diminished intracellular calcium levels ([Ca(2+)]i). Here, the therapeutic value of ursodeoxycholic acid (UDCA) was investigated. METHODS: Effect of UDCA was examined in vitro and in polycystic (PCK) rats. Hepatic cystogenesis and fibrosis, and the bile acid (BA) content were evaluated from the liver, bile, serum, and kidneys by HPLC-MS/MS. RESULTS: Chronic treatment of PCK rats with UDCA inhibits hepatic cystogenesis and fibrosis, and improves their motor behaviour. As compared to wild-type animals, PCK rats show increased BA concentration ([BA]) in liver, similar hepatic Cyp7a1 mRNA levels, and diminished [BA] in bile. Likewise, [BA] is increased in cystic fluid of PLD patients compared to their matched serum levels. In PCK rats, UDCA decreases the intrahepatic accumulation of cytotoxic BA, normalizes their diminished [BA] in bile, increases the BA secretion in bile and diminishes the increased [BA] in kidneys. In vitro, UDCA inhibits the hyperproliferation of polycystic human cholangiocytes via a PI3K/AKT/MEK/ERK1/2-dependent mechanism without affecting apoptosis. Finally, the presence of glycodeoxycholic acid promotes the proliferation of polycystic human cholangiocytes, which is inhibited by both UDCA and tauro-UDCA. CONCLUSIONS: UDCA was able to halt the liver disease of a rat model of PLD through inhibiting cystic cholangiocyte hyperproliferation and decreasing the levels of cytotoxic BA species in the liver, which suggests the use of UDCA as a potential therapeutic tool for PLD patients.
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Apoptose , Cistos/tratamento farmacológico , Hepatopatias/tratamento farmacológico , Fígado/patologia , Ácido Ursodesoxicólico/farmacologia , Animais , Ácidos e Sais Biliares/metabolismo , Ductos Biliares/metabolismo , Ductos Biliares/patologia , Cálcio/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Colagogos e Coleréticos/farmacologia , Cistos/metabolismo , Cistos/patologia , Modelos Animais de Doenças , Fígado/efeitos dos fármacos , Fígado/metabolismo , Hepatopatias/metabolismo , Hepatopatias/patologia , Ratos , Espectrometria de Massas em TandemRESUMO
Polycystic liver disease (PLD) is a member of the cholangiopathies, a group of liver diseases in which cholangiocytes, the epithelia lining of the biliary tree, are the target cells. PLDs are caused by mutations in genes involved in intracellular signaling pathways, cell cycle regulation, and ciliogenesis, among others. We previously showed that cystic cholangiocytes have abnormal cell cycle profiles and malfunctioning cilia. Because histone deacetylase 6 (HDAC6) plays an important role in both cell cycle regulation and ciliary disassembly, we examined the role of HDAC6 in hepatic cystogenesis. HDAC6 protein was increased sixfold in cystic liver tissue and in cultured cholangiocytes isolated from both PCK rats (an animal model of PLD) and humans with PLD. Furthermore, pharmacological inhibition of HDAC6 by Tubastatin-A, Tubacin, and ACY-1215 decreased proliferation of cystic cholangiocytes in a dose- and time-dependent manner, and inhibited cyst growth in three-dimensional cultures. Importantly, ACY-1215 administered to PCK rats diminished liver cyst development and fibrosis. In summary, we show that HDAC6 is overexpressed in cystic cholangiocytes both in vitro and in vivo, and its pharmacological inhibition reduces cholangiocyte proliferation and cyst growth. These data suggest that HDAC6 may represent a potential novel therapeutic target for cases of PLD.
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Cistos/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Hepatopatias/metabolismo , Anilidas/farmacologia , Anilidas/uso terapêutico , Animais , Ductos Biliares Intra-Hepáticos/citologia , Proliferação de Células , Células Cultivadas , Cílios/metabolismo , Cistos/tratamento farmacológico , Cistos/patologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Desacetilase 6 de Histona , Inibidores de Histona Desacetilases/uso terapêutico , Histona Desacetilases/efeitos dos fármacos , Humanos , Ácidos Hidroxâmicos/farmacologia , Ácidos Hidroxâmicos/uso terapêutico , Indóis/farmacologia , Indóis/uso terapêutico , Fígado/metabolismo , Fígado/patologia , Hepatopatias/tratamento farmacológico , Hepatopatias/patologia , Masculino , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Ratos , Transdução de Sinais , Fatores de TempoRESUMO
Hepatic cystogenesis in polycystic liver diseases is associated with abnormalities of cholangiocyte cilia. Given the crucial association between cilia and centrosomes, we tested the hypothesis that centrosomal defects occur in cystic cholangiocytes of rodents (Pkd2(WS25/-) mice and PCK rats) and of patients with polycystic liver diseases, contributing to disturbed ciliogenesis and cyst formation. We examined centrosomal cytoarchitecture in control and cystic cholangiocytes, the effects of centrosomal abnormalities on ciliogenesis, and the role of the cell-cycle regulator Cdc25A in centrosomal defects by depleting cholangiocytes of Cdc25A in vitro and in vivo and evaluating centrosome morphology, cell-cycle progression, proliferation, ciliogenesis, and cystogenesis. The cystic cholangiocytes had atypical centrosome positioning, supernumerary centrosomes, multipolar spindles, and extra cilia. Structurally aberrant cilia were present in cystic cholangiocytes during ciliogenesis. Depletion of Cdc25A resulted in i) a decreased number of centrosomes and multiciliated cholangiocytes, ii) an increased fraction of ciliated cholangiocytes with longer cilia, iii) a decreased proportion of cholangiocytes in G1/G0 and S phases of the cell cycle, iv) decreased cell proliferation, and v) reduced cyst growth in vitro and in vivo. Our data support the hypothesis that centrosomal abnormalities in cholangiocytes are associated with aberrant ciliogenesis and that accelerated cystogenesis is likely due to overexpression of Cdc25A, providing additional evidence that pharmacological targeting of Cdc25A has therapeutic potential in polycystic liver diseases.
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Centrossomo/metabolismo , Centrossomo/ultraestrutura , Cistos/metabolismo , Cistos/ultraestrutura , Hepatopatias/metabolismo , Fosfatases cdc25/biossíntese , Animais , Ductos Biliares/patologia , Western Blotting , Cílios/metabolismo , Cílios/ultraestrutura , Modelos Animais de Doenças , Citometria de Fluxo , Imunofluorescência , Técnicas de Inativação de Genes , Humanos , Camundongos , Microscopia Confocal , Microscopia Eletrônica , RatosRESUMO
A plasma membrane-bound G protein-coupled receptor, TGR5, that transmits bile acid signaling into a cellular response primarily via the cAMP pathway is expressed in human and rodent cholangiocytes and is localized to multiple, diverse subcellular compartments, including primary cilia. Ciliary-associated TGR5 plays an important role in cholangiocyte physiology and may contribute to a group of liver diseases referred to as the 'cholangiociliopathies', which include polycystic liver disease (PLD) and, possibly, cholangiocarcinoma and primary sclerosing cholangitis. Based on our observations that (1) ciliated and nonciliated cholangiocytes respond to TGR5 activation differently (i.e. the level of cAMP increases in nonciliated cholangiocytes but decreases in ciliated cells) and (2) hepatic cysts are derived from cholangiocytes that are characterized by both malformed cilia and increased cAMP levels, we hypothesized that TGR5-mediated cAMP signaling in cystic cholangiocytes contributes to hepatic cystogenesis. Indeed, our studies show that TGR5 is overexpressed and mislocalized in cystic cholangiocytes, and when activated by ligands, results in increased intracellular cAMP levels, cholangiocyte hyperproliferation and cyst growth. Our studies also show that genetic elimination of TGR5 in an animal model of PLD inhibits hepatic cystogenesis. Collectively, these data suggest the involvement of TGR5 in PLD and that TGR5 targeting in cystic cholangiocytes may have therapeutic potential.
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Cílios/metabolismo , AMP Cíclico/metabolismo , Cistos/genética , Cistos/metabolismo , Hepatopatias/genética , Hepatopatias/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Neoplasias dos Ductos Biliares/genética , Ductos Biliares/citologia , Colangiocarcinoma/genética , Colangite Esclerosante/genética , Células Epiteliais/metabolismo , Humanos , Transdução de SinaisRESUMO
OBJECTIVE: Polycystic liver diseases (PCLDs) are genetic disorders characterised by progressive bile duct dilatation and/or cyst development. Their pathogenesis is a consequence of hyperproliferation, hypersecretion and microRNA alterations in cholangiocytes. Here we evaluate the role of matrix metalloproteases (MMPs) in the hepatic cystogenesis of PCLDs. DESIGN: Metalloprotease activity was measured by microfluorimetric assays in normal and polycystic cholangiocyte cultures from humans and rats, and gene expression by real time quantitative PCR. The role of cytokines, oestrogens and growth factors present in the cystic fluid of PCLD patients was evaluated for MMP activity. The MMP inhibitor marimastat was examined for cystic expansion in vitro and in polycystic kidney (PCK) rats. RESULTS: Polycystic human and rat cholangiocytes displayed increased MMP activity, which was associated with increased mRNA levels of different MMPs. Interleukin (IL)-6 and IL-8, and 17ß-oestradiol, all stimulated MMP activity in human cholangiocytes. The presence of antibodies against IL-6 and/or IL-8 receptor/s inhibited baseline MMP hyperactivity of polycystic human cholangiocytes but had no effect on normal human cholangiocytes. MMP-3 was overexpressed in cystic cholangiocytes from PCLD human and PCK rat livers by immunohistochemistry. Marimastat reduced MMP hyperactivity of polycystic human and rat cholangiocytes and blocked the cystic expansion of PCK cholangiocytes cultured in three-dimensions. Chronic treatment of 8-week-old PCK rats with marimastat inhibited hepatic cystogenesis and fibrosis. CONCLUSIONS: PCLDs are associated with cholangiocyte MMP hyperactivity resulting from autocrine/paracrine stimulation by IL-6 and IL-8. Inhibition of this MMP hyperactivity with marimastat decreased hepatic cystogenesis in vitro and in an animal model of PCLD, offering a potential therapeutic tool.
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Ductos Biliares/enzimologia , Cistos/prevenção & controle , Inibidores Enzimáticos/farmacologia , Ácidos Hidroxâmicos/farmacologia , Hepatopatias/prevenção & controle , Metaloendopeptidases/antagonistas & inibidores , Animais , Ductos Biliares/patologia , Western Blotting , Técnicas de Cultura de Células , Cistos/enzimologia , Citocinas/metabolismo , Citofotometria , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Humanos , Imuno-Histoquímica , Fígado/patologia , Hepatopatias/enzimologia , Masculino , Ratos , Reação em Cadeia da Polimerase em Tempo RealAssuntos
Cistos , Hepatopatias , Humanos , Rim , Doenças Renais Policísticas , Rim Policístico Autossômico DominanteRESUMO
UNLABELLED: In polycystic liver (PLD) and kidney (PKD) diseases, increased cyclic adenosine monophosphate (cAMP) levels trigger hepatorenal cystogenesis. A reduction of the elevated cAMP by targeting somatostatin receptors (SSTRs) with octreotide (OCT; a somatostatin analog that preferentially binds to SSTR2) inhibits cyst growth. Here we compare the effects of OCT to pasireotide (PAS; a more potent somatostatin analog with broader receptor specificity) on: (1) cAMP levels, cell cycle, proliferation, and cyst expansion in vitro using cholangiocytes derived from control and PCK rats (a model of autosomal recessive PKD [ARPKD]), healthy human beings, and patients with autosomal dominant PKD (ADPKD); and (2) hepatorenal cystogenesis in vivo in PCK rats and Pkd2(WS25/-) mice (a model of ADPKD). Expression of SSTRs was assessed in control and cystic cholangiocytes of rodents and human beings. Concentrations of insulin-like growth factor 1 (IGF1) and vascular endothelial growth factor (VEGF) (both involved in indirect action of somatostatin analogs), and expression and localization of SSTRs after treatment were evaluated. We found that PAS was more potent (by 30%-45%) than OCT in reducing cAMP and cell proliferation, affecting cell cycle distribution, decreasing growth of cultured cysts in vitro, and inhibiting hepatorenal cystogenesis in vivo in PCK rats and Pkd2(WS25/-) mice. The levels of IGF1 (but not VEGF) were reduced only in response to PAS. Expression of SSTR1 and SSTR2 (but not SSTR3 and SSTR5) was decreased in cystic cholangiocytes compared to control. Although both OCT and PAS increased the immunoreactivity of SSTR2, only PAS up-regulated SSTR1; neither drug affected cellular localization of SSTRs. CONCLUSION: PAS is more effective than OCT in reducing hepatorenal cystogenesis in rodent models; therefore, it might be more beneficial for the treatment of PKD and PLD.
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Cistos/tratamento farmacológico , Hepatopatias/tratamento farmacológico , Octreotida/uso terapêutico , Doenças Renais Policísticas/tratamento farmacológico , Receptores de Somatostatina/efeitos dos fármacos , Somatostatina/análogos & derivados , Animais , Ciclo Celular/efeitos dos fármacos , AMP Cíclico/metabolismo , Humanos , Camundongos , Octreotida/metabolismo , Rim Policístico Autossômico Dominante/tratamento farmacológico , Ratos , Receptores de Somatostatina/biossíntese , Receptores de Somatostatina/metabolismo , Somatostatina/metabolismo , Somatostatina/uso terapêuticoRESUMO
Exosomes are small (30-100 nm in diameter) extracellular membrane-enclosed vesicles released by different cell types into the extracellular space or into biological fluids by exocytosis as a result of fusion of intracellular multivesicular bodies with the plasma membrane. The primary function of exosomes is intercellular communication with both beneficial (physiological) and harmful (pathological) potential outcomes. Liver cells are exosome-releasing cells as well as targets for endogenous exosomes and exosomes derived from cells of other organs. Despite limited studies on liver exosomes, initial observations suggest that these vesicles are important in liver physiology and pathophysiology. In this review, we briefly summarize the recent findings on liver exosomes, their functions and significance for novel diagnostic and therapeutic approaches.
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Exossomos/fisiologia , Hepatopatias/etiologia , Animais , Comunicação Celular , Exocitose , Exossomos/ultraestrutura , Humanos , Fígado/fisiologia , Fígado/ultraestrutura , Hepatopatias/diagnóstico , Hepatopatias/terapia , Transdução de SinaisRESUMO
TGR5, the G protein-coupled bile acid receptor that transmits bile acid signaling into a cell functional response via the intracellular cAMP signaling pathway, is expressed in human and rodent cholangiocytes. However, detailed information on the localization and function of cholangiocyte TGR5 is limited. We demonstrated that in human (H69 cells) and rat cholangiocytes, TGR5 is localized to multiple, diverse subcellular compartments, with its strongest expression on the apical plasma, ciliary, and nuclear membranes. To evaluate the relationship between ciliary TGR5 and the cholangiocyte functional response to bile acid signaling, we used a model of ciliated and nonciliated H69 cells and demonstrated that TGR5 agonists induce opposite changes in cAMP and ERK levels in cells with and without primary cilia. The cAMP level was increased in nonciliated cholangiocytes but decreased in ciliated cells. In contrast, ERK signaling was induced in ciliated cholangiocytes but suppressed in cells without cilia. TGR5 agonists inhibited proliferation of ciliated cholangiocytes but activated proliferation of nonciliated cells. The observed differential effects of TGR5 agonists were associated with the coupling of TGR5 to Gαi protein in ciliated cells and Gαs protein in nonciliated cholangiocytes. The functional responses of nonciliated and ciliated cholangiocytes to TGR5-mediated bile acid signaling may have important pathophysiological significance in cilia-related liver disorders (i.e., cholangiociliopathies), such as polycystic liver disease. In summary, TGR5 is expressed on diverse cholangiocyte compartments, including a primary cilium, and its ciliary localization determines the cholangiocyte functional response to bile acid signaling.
Assuntos
Ácidos e Sais Biliares/farmacologia , Ductos Biliares Intra-Hepáticos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Ácidos e Sais Biliares/metabolismo , Ductos Biliares Intra-Hepáticos/citologia , Linhagem Celular , Membrana Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Cílios/metabolismo , Cílios/ultraestrutura , AMP Cíclico/metabolismo , Células Epiteliais/metabolismo , Exossomos/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Camundongos , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Transporte Proteico , Ratos , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas G/agonistasRESUMO
BACKGROUND & AIMS: In polycystic kidney disease and polycystic liver disease (PLD), the normally nonproliferative hepato-renal epithelia acquire a proliferative, cystic phenotype that is linked to overexpression of cell division cycle 25 (Cdc25)A phosphatase and cell-cycle deregulation. We investigated the effects of Cdc25A inhibition in mice and rats via genetic and pharmacologic approaches. METHODS: Cdc25A(+/-) mice (which have reduced levels of Cdc25A) were cross-bred with polycystic kidney and hepatic disease 1 (Pkhd1(del2/del2)) mice (which have increased levels of Cdc25A and develop hepatic cysts). Cdc25A expression was analyzed in livers of control and polycystic kidney (PCK) rats, control and polycystic kidney 2 (Pkd2(ws25/-)) mice, healthy individuals, and patients with PLD. We examined effects of pharmacologic inhibition of Cdc25A with vitamin K3 (VK3) on the cell cycle, proliferation, and cyst expansion in vitro; hepato-renal cystogenesis in PCK rats and Pkd2(ws25/-)mice; and expression of Cdc25A and the cell-cycle proteins regulated by Cdc25A. We also examined the effects of the Cdc25A inhibitor PM-20 on hepato-renal cystogenesis in Pkd2(ws25/-) mice. RESULTS: Liver weights and hepatic and fibrotic areas were decreased by 32%-52% in Cdc25A(+/-):Pkhd1(del2/del2) mice, compared with Pkhd1(del2/del2) mice. VK3 altered the cell cycle and reduced proliferation of cultured cholangiocytes by 32%-83% and decreased growth of cultured cysts by 23%-67%. In PCK rats and Pkd2(ws25/-) mice, VK3 reduced liver and kidney weights and hepato-renal cystic and fibrotic areas by 18%-34%. PM-20 decreased hepato-renal cystogenesis in Pkd2(ws25/-) mice by 15%. CONCLUSIONS: Cdc25A inhibitors block cell-cycle progression and proliferation, reduce liver and kidney weights and cyst growth in animal models of polycystic kidney disease and PLD, and might be developed as therapeutics for these diseases.
Assuntos
Cistos/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Rim/efeitos dos fármacos , Hepatopatias/tratamento farmacológico , Fígado/efeitos dos fármacos , Rim Policístico Autossômico Recessivo/tratamento farmacológico , Vitamina K 3/farmacologia , Fosfatases cdc25/antagonistas & inibidores , Animais , Ductos Biliares Intra-Hepáticos/efeitos dos fármacos , Ductos Biliares Intra-Hepáticos/enzimologia , Ductos Biliares Intra-Hepáticos/patologia , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Cistos/enzimologia , Cistos/genética , Cistos/patologia , Modelos Animais de Doenças , Humanos , Rim/enzimologia , Rim/patologia , Fígado/enzimologia , Fígado/patologia , Hepatopatias/enzimologia , Hepatopatias/genética , Hepatopatias/patologia , Camundongos , Camundongos Knockout , Tamanho do Órgão/efeitos dos fármacos , Rim Policístico Autossômico Recessivo/enzimologia , Rim Policístico Autossômico Recessivo/genética , Rim Policístico Autossômico Recessivo/patologia , Ratos , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Canais de Cátion TRPP/genética , Canais de Cátion TRPP/metabolismo , Fatores de Tempo , Regulação para Cima , Fosfatases cdc25/deficiência , Fosfatases cdc25/genética , Fosfatases cdc25/metabolismoRESUMO
Polycystic kidney (PKD) and liver (PLD) diseases cause significant morbidity and mortality. A large body of evidence indicates that cyclic AMP plays an important role in their pathogenesis. Clinical trials of drugs that reduce cyclic AMP levels in target tissues are now in progress. Secretin may contribute to adenylyl cyclase-dependent urinary concentration and is a major agonist of adenylyl cyclase in cholangiocytes. To investigate the role of secretin in PKD and PLD, we have studied the expression of secretin and the secretin receptor in rodent models orthologous to autosomal recessive (PCK rat) and dominant (Pkd2(-/WS25) mouse) PKD; the effects of exogenous secretin administration to PCK rats, PCK rats lacking circulating vasopressin (PCK(di/di)), and Pkd2(-/WS25) mice; and the impact of a nonfunctional secretin receptor on disease development in Pkd2(-/WS25):SCTR(-/-) double mutants. Renal and hepatic secretin and secretin receptor mRNA and plasma secretin were increased in both models, and secretin receptor protein was increased in the kidneys and liver of Pkd2(-/WS25) mice. However, exogenous secretin administered subcutaneously via osmotic pumps had minimal or negligible effects and the absence of a functional secretin receptor had no influence on the severity of PKD or PLD. Therefore, it is unlikely that by itself secretin plays a significant role in the pathogenesis of PKD and/or PLD.
Assuntos
Cistos/metabolismo , Hepatopatias/metabolismo , Doenças Renais Policísticas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores dos Hormônios Gastrointestinais/metabolismo , Secretina/metabolismo , Animais , Cistos/genética , Cistos/patologia , Modelos Animais de Doenças , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Hepatopatias/genética , Hepatopatias/patologia , Camundongos , Camundongos Knockout , Doenças Renais Policísticas/genética , Doenças Renais Policísticas/patologia , Ratos , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas G/genética , Receptores dos Hormônios Gastrointestinais/genética , Secretina/genética , Secretina/farmacologiaRESUMO
Polycystic liver disease (PLD) is a group of genetic disorders characterized by progressive development of cholangiocyte-derived fluid-filled hepatic cysts. PLD is the most common manifestation of autosomal dominant and autosomal recessive polycystic kidney diseases and rarely occurs as autosomal dominant PLD. The mechanisms of PLD are a sequence of the primary (mutations in PLD-causative genes), secondary (initiation of cyst formation), and tertiary (progression of hepatic cystogenesis) interconnected molecular and cellular events in cholangiocytes. Nonsurgical, surgical, and limited pharmacological treatment options are currently available for clinical management of PLD. Substantial evidence suggests that pharmacological targeting of the signaling pathways and intracellular processes involved in the progression of hepatic cystogenesis is beneficial for PLD. Many of these targets have been evaluated in preclinical and clinical trials. In this review, we discuss the genetic, molecular, and cellular mechanisms of PLD and clinical and preclinical treatment strategies.
Assuntos
Cistos , Hepatopatias , Cistos/genética , Cistos/metabolismo , Cistos/terapia , Humanos , Hepatopatias/genética , Hepatopatias/terapia , Transdução de SinaisRESUMO
BACKGROUND & AIMS: In polycystic liver diseases, cyst formation involves cholangiocyte hyperproliferation. In polycystic kidney (PCK) rats, an animal model of autosomal-recessive polycystic kidney disease (ARPKD), decreased intracellular calcium [Ca(2+)](i) in cholangiocytes is associated with hyperproliferation. We recently showed transient receptor potential vanilloid 4 (Trpv4), a calcium-entry channel, is expressed in normal cholangiocytes and its activation leads to [Ca(2+)](i) increase. Thus, we hypothesized that pharmacologic activation of Trpv4 might reverse the hyperproliferative phenotype of PCK cholangiocytes. METHODS: Trpv4 expression was examined in liver of normal and PCK rats, normal human beings, and patients with autosomal-dominant polycystic kidney disease or ARPKD. Trpv4 activation effect on cell proliferation and cyst formation was assessed in cholangiocytes derived from normal and PCK rats. The in vivo effects of Trpv4 activation on kidney and liver cysts was analyzed in PCK rats. RESULTS: Trpv4 was overexpressed both at messenger RNA (8-fold) and protein (3-fold) levels in PCK cholangiocytes. Confocal and immunogold electron microscopy supported Trpv4 overexpression in the livers of PCK rats and ARPKD or autosomal-dominant polycystic kidney disease patients. Trpv4 activation in PCK cholangiocytes increased [Ca(2+)](i) by 30%, inhibiting cell proliferation by approximately 25%-50% and cyst growth in 3-dimensional culture (3-fold). Trpv4-small interfering RNA silencing blocked effects of Trpv4 activators by 70%. Trpv4 activation was associated with Akt phosphorylation and beta-Raf and Erk1/2 inhibition. In vivo, Trpv4 activation induced a significant decrease in renal cystic area and a nonsignificant decrease in liver cysts. CONCLUSIONS: Taken together, our in vitro and in vivo data suggest that increasing intracellular calcium by Trpv4 activation may represent a potential therapeutic approach in PKD.
Assuntos
Ductos Biliares/citologia , Rim Policístico Autossômico Recessivo/terapia , Canais de Cátion TRPV/fisiologia , Animais , Cálcio/metabolismo , Proliferação de Células , Modelos Animais de Doenças , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Humanos , Leucina/análogos & derivados , Leucina/farmacologia , Fenótipo , Ésteres de Forbol/farmacologia , Rim Policístico Autossômico Recessivo/patologia , Proteínas Proto-Oncogênicas B-raf/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologiaRESUMO
Exosomes are small extracellular vesicles that are thought to participate in intercellular communication. Recent work from our laboratory suggests that, in normal and cystic liver, exosome-like vesicles accumulate in the lumen of intrahepatic bile ducts, presumably interacting with cholangiocyte cilia. However, direct evidence for exosome-ciliary interaction is limited and the physiological relevance of such interaction remains unknown. Thus, in this study, we tested the hypothesis that biliary exosomes are involved in intercellular communication by interacting with cholangiocyte cilia and inducing intracellular signaling and functional responses. Exosomes were isolated from rat bile by differential ultracentrifugation and characterized by scanning, transmission, and immunoelectron microscopy. The exosome-ciliary interaction and its effects on ERK1/2 signaling, expression of the microRNA, miR-15A, and cholangiocyte proliferation were studied on ciliated and deciliated cultured normal rat cholangiocytes. Our results show that bile contains vesicles identified as exosomes by their size, characteristic "saucer-shaped" morphology, and specific markers, CD63 and Tsg101. When NRCs were exposed to isolated biliary exosomes, the exosomes attached to cilia, inducing a decrease of the phosphorylated-to-total ERK1/2 ratio, an increase of miR-15A expression, and a decrease of cholangiocyte proliferation. All these effects of biliary exosomes were abolished by the pharmacological removal of cholangiocyte cilia. Our findings suggest that bile contains exosomes functioning as signaling nanovesicles and influencing intracellular regulatory mechanisms and cholangiocyte proliferation through interaction with primary cilia.