RESUMEN
Chronic cholestasis results from bile secretory defects or impaired bile flow with few effective medical therapies available. Thyroid hormone triiodothyronine and synthetic thyroid hormone receptor agonists, such as sobetirome (GC-1), are known to impact lipid and bile acid (BA) metabolism and induce hepatocyte proliferation downstream of Wnt/ß-catenin signaling after surgical resection; however, these drugs have yet to be studied as potential therapeutics for cholestatic liver disease. Herein, GC-1 was administered to ATP binding cassette subfamily B member 4 (Abcb4-/-; Mdr2-/-) knockout (KO) mice, a sclerosing cholangitis model. KO mice fed GC-1 diet for 2 and 4 weeks had decreased serum alkaline phosphatase but increased serum transaminases compared with KO alone. KO mice on GC-1 also had higher levels of total liver BA due to alterations in expression of BA detoxification, transport, and synthesis genes, with the net result being retention of BA in the hepatocytes. Interestingly, GC-1 does not induce hepatocyte proliferation or Wnt/ß-catenin signaling in KO mice, likely a result of decreased thyroid hormone receptor ß expression without Mdr2. Therefore, although GC-1 treatment induces a mild protection against biliary injury in the early stages of treatment, it comes at the expense of hepatocyte injury and is suboptimal because of lower expression of thyroid hormone receptor ß. Thus, thyromimetics may have limited therapeutic benefits in treating cholestatic liver disease.
Asunto(s)
Acetatos/farmacología , Ácidos y Sales Biliares/metabolismo , Colestasis Intrahepática , Hepatocitos/efectos de los fármacos , Fenoles/farmacología , Animales , Modelos Animales de Enfermedad , Ratones , Ratones NoqueadosRESUMEN
BACKGROUND AND AIMS: The Wnt/ß-catenin signaling pathway has a well-described role in liver pathobiology. Its suppression was recently shown to decrease bile acid (BA) synthesis, thus preventing the development of cholestatic liver injury and fibrosis after bile duct ligation (BDL). APPROACH AND RESULTS: To generalize these observations, we suppressed ß-catenin in Mdr2 knockout (KO) mice, which develop sclerosing cholangitis due to regurgitation of BA from leaky ducts. When ß-catenin was knocked down (KD) in KO for 2 weeks, hepatic and biliary injury were exacerbated in comparison to KO given placebo, as shown by serum biochemistry, ductular reaction, inflammation, and fibrosis. Simultaneously, KO/KD livers displayed increased oxidative stress and senescence and an impaired regenerative response. Although the total liver BA levels were similar between KO/KD and KO, there was significant dysregulation of BA transporters and BA detoxification/synthesis enzymes in KO/KD compared with KO alone. Multiphoton intravital microscopy revealed a mixing of blood and bile in the sinusoids, and validated the presence of increased serum BA in KO/KD mice. Although hepatocyte junctions were intact, KO/KD livers had significant canalicular defects, which resulted from loss of hepatocyte polarity. Thus, in contrast to the protective effect of ß-catenin KD in BDL model, ß-catenin KD in Mdr2 KO aggravated rather than alleviated injury by interfering with expression of BA transporters, hepatocyte polarity, canalicular structure, and the regenerative response. CONCLUSIONS: The resulting imbalance between ongoing injury and restitution led to worsening of the Mdr2 KO phenotype, suggesting caution in targeting ß-catenin globally for all cholestatic conditions.
Asunto(s)
Colangitis Esclerosante/complicaciones , Colangitis Esclerosante/genética , Colestasis/etiología , Colestasis/metabolismo , Vía de Señalización Wnt , beta Catenina/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Animales , Ácidos y Sales Biliares/metabolismo , Conductos Biliares/patología , Colestasis/genética , Modelos Animales de Enfermedad , Hepatocitos/metabolismo , Hepatocitos/patología , Uniones Intercelulares/metabolismo , Uniones Intercelulares/patología , Ratones Noqueados , Miembro 4 de la Subfamilia B de Casete de Unión a ATPRESUMEN
BACKGROUND & AIMS: Porphyrias result from anomalies of heme biosynthetic enzymes and can lead to cirrhosis and hepatocellular cancer. In mice, these diseases can be modeled by administration of a diet containing 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC), which causes accumulation of porphyrin intermediates, resulting in hepatobiliary injury. Wnt/ß-catenin signaling has been shown to be a modulatable target in models of biliary injury; thus, we investigated its role in DDC-driven injury. METHODS: ß-Catenin (Ctnnb1) knockout (KO) mice, Wnt co-receptor KO mice, and littermate controls were fed a DDC diet for 2â¯weeks. ß-Catenin was exogenously inhibited in hepatocytes by administering ß-catenin dicer-substrate RNA (DsiRNA), conjugated to a lipid nanoparticle, to mice after DDC diet and then weekly for 4â¯weeks. In all experiments, serum and livers were collected; livers were analyzed by histology, western blotting, and real-time PCR. Porphyrin was measured by fluorescence, quantification of polarized light images, and liquid chromatography-mass spectrometry. RESULTS: DDC-fed mice lacking ß-catenin or Wnt signaling had decreased liver injury compared to controls. Exogenous mice that underwent ß-catenin suppression by DsiRNA during DDC feeding also showed less injury compared to control mice receiving lipid nanoparticles. Control livers contained extensive porphyrin deposits which were largely absent in mice lacking ß-catenin signaling. Notably, we identified a network of key heme biosynthesis enzymes that are suppressed in the absence of ß-catenin, preventing accumulation of toxic protoporphyrins. Additionally, mice lacking ß-catenin exhibited fewer protein aggregates, improved proteasomal activity, and reduced induction of autophagy, all contributing to protection from injury. CONCLUSIONS: ß-Catenin inhibition, through its pleiotropic effects on metabolism, cell stress, and autophagy, represents a novel therapeutic approach for patients with porphyria. LAY SUMMARY: Porphyrias are disorders resulting from abnormalities in the steps that lead to heme production, which cause build-up of toxic by-products called porphyrins. Liver is commonly either a source or a target of excess porphyrins, and complications can range from minor abnormalities to liver failure. In this report, we inhibited Wnt/ß-catenin signaling in an experimental model of porphyria, which resulted in decreased liver injury. Targeting ß-catenin affected multiple components of the heme biosynthesis pathway, thus preventing build-up of porphyrin intermediates. Our study suggests that drugs inhibiting ß-catenin activity could reduce the amount of porphyrin accumulation and help alleviate symptoms in patients with porphyria.
Asunto(s)
Hepatocitos/metabolismo , Cirrosis Hepática/metabolismo , Porfirias/complicaciones , Porfirinas/metabolismo , beta Catenina/metabolismo , Animales , Proliferación Celular , Células Cultivadas , Modelos Animales de Enfermedad , Hepatocitos/patología , Inmunohistoquímica , Cirrosis Hepática/etiología , Cirrosis Hepática/patología , Masculino , Ratones , Ratones NoqueadosRESUMEN
BACKGROUND & AIMS: Liver fibrosis, hepatocellular necrosis, inflammation, and proliferation of liver progenitor cells are features of chronic liver injury. Mouse models have been used to study the end-stage pathophysiology of chronic liver injury. However, little is known about differences in the mechanisms of liver injury among different mouse models because of our inability to visualize the progression of liver injury in vivo in mice. We developed a method to visualize bile transport and blood-bile barrier (BBlB) integrity in live mice. METHODS: C57BL/6 mice were fed a choline-deficient, ethionine-supplemented (CDE) diet or a diet containing 0.1% 3,5-diethoxycarbonyl-1, 4-dihydrocollidine (DDC) for up to 4 weeks to induce chronic liver injury. We used quantitative liver intravital microscopy (qLIM) for real-time assessment of bile transport and BBlB integrity in the intact livers of the live mice fed the CDE, DDC, or chow (control) diets. Liver tissues were collected from mice and analyzed by histology, immunohistochemistry, real-time polymerase chain reaction, and immunoblots. RESULTS: Mice with liver injury induced by a CDE or a DDC diet had breaches in the BBlB and impaired bile secretion, observed by qLIM compared with control mice. Impaired bile secretion was associated with reduced expression of several tight-junction proteins (claudins 3, 5, and 7) and bile transporters (NTCP, OATP1, BSEP, ABCG5, and ABCG8). A prolonged (2-week) CDE, but not DDC, diet led to re-expression of tight junction proteins and bile transporters, concomitant with the reestablishment of BBlB integrity and bile secretion. CONCLUSIONS: We used qLIM to study chronic liver injury, induced by a choline-deficient or DDC diet, in mice. Progression of chronic liver injury was accompanied by loss of bile transporters and tight junction proteins.
Asunto(s)
Bilis/metabolismo , Enfermedad Hepática Crónica Inducida por Sustancias y Drogas/metabolismo , Hepatocitos/metabolismo , Hígado/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Uniones Estrechas/metabolismo , Animales , Transporte Biológico , Enfermedad Hepática Crónica Inducida por Sustancias y Drogas/sangre , Enfermedad Hepática Crónica Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Crónica Inducida por Sustancias y Drogas/patología , Deficiencia de Colina/complicaciones , Claudinas/metabolismo , Modelos Animales de Enfermedad , Etionina , Hepatocitos/patología , Cinética , Hígado/patología , Ratones Endogámicos C57BL , Permeabilidad , Piridinas , Uniones Estrechas/patologíaRESUMEN
Cholestatic liver diseases result from impaired bile flow and are characterized by inflammation, atypical ductular proliferation, and fibrosis. The Wnt/ß-catenin pathway plays a role in bile duct development, yet its role in cholestatic injury remains indeterminate. Liver-specific ß-catenin knockout mice and wild-type littermates were subjected to cholestatic injury through bile duct ligation or short-term exposure to 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet. Intriguingly, knockout mice exhibit a dramatic protection from liver injury, fibrosis, and atypical ductular proliferation, which coincides with significantly decreased total hepatic bile acids (BAs). This led to the discovery of a role for ß-catenin in regulating BA synthesis and transport through regulation of farnesoid X receptor (FXR) activation. We show that ß-catenin functions as both an inhibitor of nuclear translocation and a nuclear corepressor through formation of a physical complex with FXR. Loss of ß-catenin expedited FXR nuclear localization and FXR/retinoic X receptor alpha association, culminating in small heterodimer protein promoter occupancy and activation in response to BA or FXR agonist. Conversely, accumulation of ß-catenin sequesters FXR, thus inhibiting its activation. Finally, exogenous suppression of ß-catenin expression during cholestatic injury reduces ß-catenin/FXR complex activation of FXR to decrease total BA and alleviate hepatic injury. CONCLUSION: We have identified an FXR/ß-catenin interaction whose modulation through ß-catenin suppression promotes FXR activation and decreases hepatic BAs, which may provide unique therapeutic opportunities in cholestatic liver diseases. (Hepatology 2018;67:955-971).
Asunto(s)
Ácidos y Sales Biliares/metabolismo , Colestasis/metabolismo , Hígado/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , beta Catenina/metabolismo , Animales , Hígado/patología , Ratones , Ratones Noqueados , Transducción de SeñalRESUMEN
An important role for ß-catenin in regulating p65 (a subunit of NF-κB) during acute liver injury has recently been elucidated through use of conditional ß-catenin knockout mice, which show protection from apoptosis through increased activation of p65. Thus, we hypothesized that the p65/ß-catenin complex may play a role in regulating processes such as cell proliferation during liver regeneration. We show through in vitro and in vivo studies that the p65/ß-catenin complex is regulated through the TNF-α pathway and not through Wnt signaling. However, this complex is unchanged after partial hepatectomy (PH), despite increased p65 and ß-catenin nuclear translocation as well as cyclin D1 activation. We demonstrate through both in vitro silencing experiments and chromatin immunoprecipitation after PH that ß-catenin, and not p65, regulates cyclin D1 expression. Conversely, using reporter mice we show p65 is activated exclusively in the nonparenchymal (NPC) compartment during liver regeneration. Furthermore, stimulation of macrophages by TNF-α induces activation of NF-κB and subsequent secretion of Wnts essential for ß-catenin activation in hepatocytes. Thus, we show that ß-catenin and p65 are activated in separate cellular compartments during liver regeneration, with p65 activity in NPCs contributing to the activation of hepatocyte ß-catenin, cyclin D1 expression, and subsequent proliferation.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Regulación de la Expresión Génica , Regeneración Hepática , Hígado/metabolismo , Factor de Transcripción ReIA/metabolismo , beta Catenina/metabolismo , Transporte Activo de Núcleo Celular , Animales , Línea Celular Tumoral , Proliferación Celular , Ciclina D1/metabolismo , Hepatectomía , Hepatocitos/metabolismo , Humanos , Macrófagos/metabolismo , Ratones , Ratones Noqueados , Ratones Transgénicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Células RAW 264.7 , Factor de Transcripción ReIA/genética , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/genéticaRESUMEN
BACKGROUND: Cholestasis is an intractable liver disorder that results from impaired bile flow. We have previously shown that the Wnt/ß-catenin signaling pathway regulates the progression of cholestatic liver disease through multiple mechanisms, including bile acid metabolism and hepatocyte proliferation. To further explore the impact of these functions during intrahepatic cholestasis, we exposed mice to a xenobiotic that causes selective biliary injury. METHODS: α-naphthylisothiocyanate (ANIT) was administered to liver-specific knockout (KO) of ß-catenin and wild-type mice in the diet. Mice were killed at 6 or 14 days to assess the severity of cholestatic liver disease, measure the expression of target genes, and perform biochemical analyses. RESULTS: We found that the presence of ß-catenin was protective against ANIT, as KO mice had a significantly lower survival rate than wild-type mice. Although serum markers of liver damage and total bile acid levels were similar between KO and wild-type mice, the KO had minor histological abnormalities, such as sinusoidal dilatation, concentric fibrosis around ducts, and decreased inflammation. Notably, both total glutathione levels and expression of glutathione-S-transferases, which catalyze the conjugation of ANIT to glutathione, were significantly decreased in KO after ANIT. Nuclear factor erythroid-derived 2-like 2, a master regulator of the antioxidant response, was activated in KO after ANIT as well as in a subset of patients with primary sclerosing cholangitis lacking activated ß-catenin. Despite the activation of nuclear factor erythroid-derived 2-like 2, KO livers had increased lipid peroxidation and cell death, which likely contributed to mortality. CONCLUSIONS: Loss of ß-catenin leads to increased cellular injury and cell death during cholestasis through failure to neutralize oxidative stress, which may contribute to the pathology of this disease.
Asunto(s)
1-Naftilisotiocianato , Colestasis Intrahepática , Glutatión , Ratones Noqueados , Estrés Oxidativo , beta Catenina , Animales , beta Catenina/metabolismo , Ratones , Glutatión/metabolismo , Colestasis Intrahepática/metabolismo , Hígado/metabolismo , Hígado/patología , Ácidos y Sales Biliares/metabolismo , Humanos , Masculino , Modelos Animales de EnfermedadRESUMEN
BACKGROUND & AIMS: ß-Catenin, the effector molecule of the Wnt signaling pathway, has been shown to play a crucial role in bile acid homeostasis through direct inhibition of farnesoid X receptor (FXR), which has pleiotropic effects on bile acid homeostasis. We hypothesize that simultaneous suppression of ß-catenin signaling and activation of FXR in a mouse model of cholestasis will reduce injury and biliary fibrosis through inhibition of bile acid synthesis. METHODS: To induce cholestasis, we performed bile duct ligation (BDL) on wild-type male mice. Eight hours after surgery, mice received FXR agonists obeticholic acid, tropifexor, or GW-4064 or Wnt inhibitor Wnt-C59. Severity of cholestatic liver disease and expression of target genes were evaluated after either 5 days or 12 days of treatment. RESULTS: We found that although the FXR agonists worsened BDL-induced injury and necrosis after 5 days, Wnt-C59 did not. After 12 days of BDL, Wnt-C59 treatment, but not GW-4064 treatment, reduced both the number of infarcts and the number of inflammatory cells in liver. RNA sequencing analysis of whole livers revealed a notable suppression of nuclear factor kappa B signaling when Wnt signaling is inhibited. We then analyzed transcriptomic data to identify a cholangiocyte-specific signature in our model and demonstrated that Wnt-C59-treated livers were enriched for genes expressed in quiescent cholangiocytes, whereas genes expressed in activated cholangiocytes were enriched in BDL alone. A similar decrease in biliary injury and inflammation occurred in Mdr2 KO mice treated with Wnt-C59. CONCLUSIONS: Inhibiting Wnt signaling suppresses cholangiocyte activation and disrupts the nuclear factor kappa B-dependent inflammatory axis, reducing cholestatic-induced injury.
Asunto(s)
Colestasis , Vía de Señalización Wnt , Masculino , Animales , Ratones , beta Catenina , FN-kappa B , Ácidos y Sales BiliaresRESUMEN
Sox11 is a high-mobility group (HMG)-containing transcription factor that is significantly elevated in peripheral neurons in response to nerve injury. In vitro and in vivo studies support a central role for Sox11 in adult neuron growth and survival following injury. Brain-derived neurotrophic factor (BDNF) is a pleiotropic growth factor that has effects on neuronal survival, differentiation, synaptic plasticity, and regeneration. BDNF transcription is elevated in the dorsal root ganglia (DRG) following nerve injury in parallel with Sox11, allowing for the possible regulation by Sox11. To begin to assess the possible influence of Sox11, we used reverse transcriptase PCR assays to determine the relative expression of the nine (I-IXa) noncoding exons and one coding exon (exon IX) of the BDNF gene after sciatic nerve axotomy in the mouse. Exons with upstream promoter regions containing the Sox binding motif 5'-AACAAAG-3' (I, IV, VII, and VIII) were increased at 1 or 3 days following axotomy. Exons 1 and IV showed the greatest increase, and only exon 1 remained elevated at 3 days. Luciferase assays showed that Sox11 could activate the most highly regulated exons, I and IV, and that this activation was reduced by mutation of putative Sox binding sites. Exon expression in injured DRG neurons had some overlap with Neuro2a cells that overexpress Sox11, showing elevation in exon IV and VII transcripts. These findings indicate cell type and contextual specificity of Sox11 in modulation of BDNF transcription.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/metabolismo , Exones/fisiología , Ganglios Espinales/metabolismo , Regulación de la Expresión Génica/fisiología , Neuronas/metabolismo , Factores de Transcripción SOXB1/fisiología , Animales , Axotomía , Factor Neurotrófico Derivado del Encéfalo/genética , Línea Celular Tumoral , Biología Computacional , Modelos Animales de Enfermedad , Ganglios Espinales/patología , Regulación de la Expresión Génica/genética , Vectores Genéticos/fisiología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , VIH/genética , Luciferasas/genética , Luciferasas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Mutagénesis Sitio-Dirigida , Neuroblastoma/patología , Factores de Transcripción SOXB1/genética , Neuropatía Ciática/patología , Factores de Tiempo , Transducción Genética , TransfecciónRESUMEN
Primary sclerosing cholangitis (PSC) is a rare, chronic, cholestatic liver disease characterized by progressive inflammation and fibrosis of the bile ducts. We have previously demonstrated the importance of Wnt/ß-catenin signaling in mouse models of PSC. In this study, we wished to determine the clinical relevance of ß-catenin localization in patient samples. In livers explanted from patients diagnosed with PSC, the majority (12/16; 75%) lacked ß-catenin protein expression. Biopsies from patients post-transplant were classified as recurrent or non-recurrent based on pathology reports and then scored for ß-catenin activation as a function of immunohistochemical localization. Despite lack of statistical significance, patients with recurrent primary disease (n = 11) had a greater percentage of samples with nuclear, transcriptionally active ß-catenin (average 58.8%) than those with no recurrence (n = 10; 40.53%), while non-recurrence is correlated with ß-catenin staining at the cell surface (average 52.63% for non-recurrent vs. 27.34% for recurrent), as determined by three different methods of analysis. ß-catenin score and years-to-endpoint are both strongly associated with recurrence status (p = 0.017 and p = 0.00063, respectively). Finally, there was significant association between higher ß-catenin score and increased alkaline phosphatase, a marker of biliary injury and disease progression. Thus, ß-catenin expression and activation changes during the progression of PSC, and its localization may be a useful prognostic tool for predicting recurrence of this disease.
Asunto(s)
Colangitis Esclerosante/metabolismo , Hígado/metabolismo , beta Catenina/metabolismo , Fosfatasa Alcalina/metabolismo , Ácidos y Sales Biliares/metabolismo , Biomarcadores/metabolismo , Colangitis Esclerosante/patología , Colangitis Esclerosante/cirugía , Progresión de la Enfermedad , Humanos , Hígado/patología , Hígado/cirugía , Trasplante de Hígado , Valor Predictivo de las Pruebas , Recurrencia , Estudios Retrospectivos , Factores de Tiempo , Resultado del TratamientoRESUMEN
We previously identified an up-regulation of specific Wnt proteins in the cholangiocyte compartment during cholestatic liver injury and found that mice lacking Wnt secretion from hepatocytes and cholangiocytes showed fewer proliferating cholangiocytes and high mortality in response to a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet, a murine model of primary sclerosing cholangitis. In vitro studies demonstrated that Wnt7b, one of the Wnts up-regulated during cholestasis, induces proliferation of cholangiocytes in an autocrine manner and increases secretion of proinflammatory cytokines. We hypothesized that loss of Wnt7b may exacerbate some of the complications of cholangiopathies by decreasing the ability of bile ducts to induce repair. Wnt7b-flox mice were bred with Krt19-cre mice to deplete Wnt7b expression in only cholangiocytes (CC) or with albumin-Cre mice to delete Wnt7b expression in both hepatocytes and cholangiocytes (HC + CC). These mice were placed on a DDC diet for 1 month then killed for evaluation. Contrary to our expectations, we found that mice lacking Wnt7b from CC and HC + CC compartments had improved biliary injury, decreased cellular senescence, and lesser bile acid accumulation after DDC exposure compared to controls, along with decreased expression of inflammatory cytokines. Although Wnt7b knockout (KO) resulted in fewer proliferating cholangiocytes, CC and HC + CC KO mice on a DDC diet also had more hepatocytes expressing cholangiocyte markers compared to wild-type mice on a DDC diet, indicating that Wnt7b suppression promotes hepatocyte reprogramming. Conclusion: Wnt7b induces a proproliferative proinflammatory program in cholangiocytes, and its loss is compensated for by conversion of hepatocytes to a biliary phenotype during cholestatic injury.
Asunto(s)
Conductos Biliares/citología , Proliferación Celular/genética , Colestasis/genética , Proteínas Proto-Oncogénicas/deficiencia , Proteínas Wnt/deficiencia , Animales , Ácidos y Sales Biliares/metabolismo , Senescencia Celular/genética , Modelos Animales de Enfermedad , Hepatocitos/metabolismo , Ratones , Ratones NoqueadosRESUMEN
We have recently shown that loss of ß-catenin prevents the development of cholestatic liver injury and fibrosis after bile duct ligation (BDL) due to loss of the inhibitory farnesoid X receptor (FXR)/ß-catenin complex, which results in decreased hepatic bile acids (BAs) through activation of FXR. To further understand the role of Wnt/ß-catenin signaling in regulating BA metabolism and cholestasis, we performed BDL on mice in which hepatocyte Wnt signaling is deficient but ß-catenin is intact (low-density lipoprotein receptor-related protein [LRP]5/6 knockout [DKO]) as well as mice that have enhanced hepatocyte ß-catenin expression (serine 45 mutated to aspartic acid [S45D] transgenic [TG] mice). Despite decreased biliary injury after BDL, hepatic injury, fibrosis, and inflammation were comparable in DKO and wild-type (WT) mice. Notably, the FXR/ß-catenin complex was maintained in DKO livers after BDL, coincident with significantly elevated hepatic BA levels. Similarly, TG mice did not display accelerated injury or increased mortality despite overexpression of ß-catenin. There was no augmentation of FXR/ß-catenin association in TG livers; this resulted in equivalent hepatic BAs in WT and TG mice after BDL. Finally, we analyzed the effect of BDL on ß-catenin activity and identified an increase in periportal cytoplasmic stabilization and association with T-cell factor 4 that correlated with increased expression of distinct downstream target genes. Conclusion: Localization of ß-catenin and expression of Wnt-regulated genes were altered in liver after BDL; however, neither elimination of Wnt/ß-catenin signaling nor overexpression of ß-catenin in hepatocytes significantly impacted the phenotype or progression of BA-driven cholestatic injury.
RESUMEN
Nerve growth factor (NGF) has been implicated as an effector of inflammatory pain because it sensitizes primary afferents to noxious thermal, mechanical, and chemical [e.g., capsaicin, a transient receptor potential vanilloid receptor 1 (TRPV1) agonist] stimuli and because NGF levels increase during inflammation. Here, we report the ability of glial cell line-derived neurotrophic factor (GDNF) family members artemin, neurturin and GDNF to potentiate TRPV1 signaling and to induce behavioral hyperalgesia. Analysis of capsaicin-evoked Ca2+ transients in dissociated mouse dorsal root ganglion (DRG) neurons revealed that a 7 min exposure to GDNF, neurturin, or artemin potentiated TRPV1 function at doses 10-100 times lower than NGF. Moreover, GDNF family members induced capsaicin responses in a subset of neurons that were previously insensitive to capsaicin. Using reverse transcriptase-PCR, we found that artemin mRNA was profoundly upregulated in response to inflammation induced by hindpaw injection of complete Freund's adjuvant (CFA): artemin expression increased 10-fold 1 d after CFA injection, whereas NGF expression doubled by day 7. No increase was seen in neurturin or GDNF. A corresponding increase in mRNA for the artemin coreceptor GFRalpha3 (for GDNF family receptor alpha) was seen in DRG, and GFRalpha3 immunoreactivity was widely colocalized with TRPV1 in epidermal afferents. Finally, hindpaw injection of artemin, neurturin, GDNF, or NGF produced acute thermal hyperalgesia that lasted up to 4 h; combined injection of artemin and NGF produced hyperalgesia that lasted for 6 d. These results indicate that GDNF family members regulate the sensitivity of thermal nociceptors and implicate artemin in particular as an important effector in inflammatory hyperalgesia.
Asunto(s)
Factor Neurotrófico Derivado de la Línea Celular Glial/fisiología , Calor , Hiperalgesia/etiología , Familia de Multigenes/fisiología , Nociceptores/fisiología , Animales , Capsaicina/farmacología , Células Cultivadas , Sinergismo Farmacológico , Ganglios Espinales/metabolismo , Factor Neurotrófico Derivado de la Línea Celular Glial/farmacología , Sustancias de Crecimiento/metabolismo , Sustancias de Crecimiento/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Factor de Crecimiento Nervioso/farmacología , Proteínas del Tejido Nervioso/metabolismo , Neuronas Aferentes/metabolismo , Neurturina/metabolismo , Receptores de Superficie Celular/metabolismo , Piel/inervación , Canales Catiónicos TRPV/metabolismo , Factores de Tiempo , Distribución TisularRESUMEN
Artemin, a neuronal survival factor in the glial cell line-derived neurotrophic factor family, binds the glycosylphosphatidylinositol-anchored protein GFRalpha3 and the receptor tyrosine kinase Ret. Expression of the GFRalpha3 receptor is primarily restricted to the peripheral nervous system and is found in a subpopulation of nociceptive sensory neurons of the dorsal root ganglia (DRGs) that coexpress the Ret and TrkA receptor tyrosine kinases and the thermosensitive channel TRPV1. To determine how artemin affects sensory neuron properties, transgenic mice that overexpress artemin in skin keratinocytes (ART-OE mice) were analyzed. Expression of artemin caused a 20.5% increase in DRG neuron number and increased the level of mRNA encoding GFRalpha3, TrkA, TRPV1, and the putative noxious cold-detecting channel TRPA1. Nearly all GFRalpha3-positive neurons expressed TRPV1 immunoreactivity, and most of these neurons were also positive for TRPA1. Interestingly, acid-sensing ion channel (ASIC) 1, 2a, 2b, and 3 mRNAs were decreased in the DRG, and this reduction was strongest in females. Analysis of sensory neuron physiological properties using an ex vivo preparation showed that cutaneous C-fiber nociceptors of ART-OE mice had reduced heat thresholds and increased firing rates in response to a heat ramp. No change in mechanical threshold was detected. Behavioral testing of ART-OE mice showed that they had increased sensitivity to both heat and noxious cold. These results indicate that the level of artemin in the skin modulates gene expression and response properties of afferents that project to the skin and that these changes lead to behavioral sensitivity to both hot and cold stimuli.
Asunto(s)
Conducta Animal/fisiología , Proteínas del Tejido Nervioso/metabolismo , Neuronas Aferentes/metabolismo , Piel/inervación , Canales Catiónicos TRPV/metabolismo , Temperatura , Canales de Potencial de Receptor Transitorio/metabolismo , Canales Iónicos Sensibles al Ácido , Animales , Biomarcadores/metabolismo , Capsaicina/farmacología , Recuento de Células , Frío , Femenino , Ganglios Sensoriales/citología , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Calor , Hipertrofia , Masculino , Mecanorreceptores/fisiología , Proteínas de la Membrana/genética , Ratones , Ratones Transgénicos , Fibras Nerviosas Amielínicas/fisiología , Proteínas del Tejido Nervioso/genética , Neuronas Aferentes/citología , Neuronas Aferentes/efectos de los fármacos , Neuronas Aferentes/patología , ARN Mensajero/metabolismo , Caracteres Sexuales , Piel/metabolismo , Canales de Sodio/genética , Canal Catiónico TRPA1 , Canales Catiónicos TRPV/genética , Termorreceptores/fisiologíaRESUMEN
Stem cell-based therapy of retinal degenerative conditions is a promising modality to treat blindness, but requires new strategies to improve the number of functionally integrating cells. Grafting semidifferentiated retinal tissue rather than progenitors allows preservation of tissue structure and connectivity in retinal grafts, mandatory for vision restoration. Using human embryonic stem cells (hESCs), we derived retinal tissue growing in adherent conditions consisting of conjoined neural retina and retinal pigment epithelial (RPE) cells and evaluated cell fate determination and maturation in this tissue. We found that deriving such tissue in adherent conditions robustly induces all eye field genes (RX, PAX6, LHX2, SIX3, SIX6) and produces four layers of pure populations of retinal cells: RPE (expressing NHERF1, EZRIN, RPE65, DCT, TYR, TYRP, MITF, PMEL), early photoreceptors (PRs) (coexpressing CRX and RCVRN), inner nuclear layer neurons (expressing CALB2), and retinal ganglion cells [RGCs, expressing BRN3B and Neurofilament (NF) 200]. Furthermore, we found that retinal progenitors divide at the apical side of the hESC-derived retinal tissue (next to the RPE layer) and then migrate toward the basal side, similar to that found during embryonic retinogenesis. We detected synaptogenesis in hESC-derived retinal tissue, and found neurons containing many synaptophysin-positive boutons within the RGC and PR layers. We also observed long NF200-positive axons projected by RGCs toward the apical side. Whole-cell recordings demonstrated that putative amacrine and/or ganglion cells exhibited electrophysiological responses reminiscent of those in normal retinal neurons. These responses included voltage-gated Na(+) and K(+) currents, depolarization-induced spiking, and responses to neurotransmitter receptor agonists. Differentiation in adherent conditions allows generation of long and flexible pieces of 3D retinal tissue suitable for isolating transplantable slices of tissue for retinal replacement therapies.
Asunto(s)
Células Madre Embrionarias/citología , Neuronas Retinianas/citología , Epitelio Pigmentado de la Retina/citología , Ingeniería de Tejidos , Potenciales de Acción , Células Cultivadas , Células Madre Embrionarias/metabolismo , Humanos , Neurogénesis , Potasio/metabolismo , Neuronas Retinianas/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Sodio/metabolismo , Sinapsis/metabolismo , Sinapsis/fisiología , Sinaptofisina/genética , Sinaptofisina/metabolismoRESUMEN
CFTR is an integral transmembrane glycoprotein and a cAMP-activated Cl(-) channel. Mutations in the CFTR gene lead to Cystic Fibrosis (CF)-an autosomal recessive disease with majority of the morbidity and mortality resulting from airway infection, inflammation, and fibrosis. The most common disease-associated mutation in the CFTR gene-deletion of Phe508 (ΔF508) leads to a biosynthetic processing defect of CFTR. Correction of the defect and delivery of ΔF508-CFTR to the cell surface has been highly anticipated as a disease modifying therapy. Compared to promising results in cultured cell this approach was much less effective in CF patients in an early clinical trial. Although the cause of failure to rescue ΔF508-CFTR in the clinical trial has not been determined, presence of factor(s) that interfere with the rescue in vivo could be considered. The cytokine TGF-ß1 is frequently elevated in CF patients. TGF-ß1 has pleiotropic effects in different disease models and genetic backgrounds and little is known about TGF-ß1 effects on CFTR in human airway epithelial cells. Moreover, there are no published studies examining TGF-ß1 effects on the functional rescue of ΔF508-CFTR. Here we found that TGF-ß1 inhibits CFTR biogenesis by reducing mRNA levels and protein abundance in primary differentiated human bronchial epithelial (HBE) cells from non-CF individuals. TGF-ß1 inhibits CFTR biogenesis without compromising the epithelial phenotype or integrity of HBE cells. TGF-ß1 also inhibits biogenesis and impairs the functional rescue of ΔF508-CFTR in HBE cells from patients homozygous for the ΔF508 mutation. Our data indicate that activation of TGF-ß1 signaling may inhibit CFTR function in non-CF individuals and may interfere with therapies directed at correcting the processing defect of ΔF508-CFTR in CF patients.
Asunto(s)
Diferenciación Celular , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Células Epiteliales/efectos de los fármacos , Factor de Crecimiento Transformador beta1/farmacología , Western Blotting , Bronquios/citología , Membrana Celular/metabolismo , Células Cultivadas , Cloruros/metabolismo , Fibrosis Quística/genética , Fibrosis Quística/metabolismo , Fibrosis Quística/patología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Células Epiteliales/metabolismo , Expresión Génica/efectos de los fármacos , Humanos , Mutación , Proteínas Serina-Treonina Quinasas/metabolismo , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The ability of adult peripheral sensory neurons to undergo functional and anatomical recovery following nerve injury is due in part to successful activation of transcriptional regulatory pathways. Previous in vitro evidence had suggested that the transcription factor Sox11, a HMG-domain containing protein that is highly expressed in developing sensory neurons, is an important component of this regenerative transcriptional control program. To further test the role of Sox11 in an in vivo system, we developed a new approach to specifically target small interfering RNAs (siRNAs) conjugated to the membrane permeable molecule Penetratin to injured sensory afferents. Injection of Sox11 siRNAs into the mouse saphenous nerve caused a transient knockdown of Sox11 mRNA that transiently inhibited in vivo regeneration. Electron microscopic level analysis of Sox11 RNAi-injected nerves showed that regeneration of myelinated and unmyelinated axons was inhibited. Nearly all neurons in ganglia of crushed nerves that were Sox11 immunopositive showed colabeling for the stress and injury-associated activating transcription factor 3 (ATF3). In addition, treatment with Sox11 siRNAs in vitro and in vivo caused a transcriptional and translational level reduction in ATF3 expression. These anatomical and expression data support an intrinsic role for Sox11 in events that underlie successful regeneration following peripheral nerve injury.
Asunto(s)
Regeneración Nerviosa , Nervios Periféricos/fisiología , Factores de Transcripción SOXC/metabolismo , Células Receptoras Sensoriales/metabolismo , Factor de Transcripción Activador 3/metabolismo , Animales , Proteínas Portadoras/metabolismo , Línea Celular , Péptidos de Penetración Celular , Células Cultivadas , Regulación hacia Abajo , Ganglios Espinales/metabolismo , Técnicas de Silenciamiento del Gen , Masculino , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/metabolismo , ARN Interferente Pequeño , Factores de Transcripción SOXC/genética , Células Receptoras Sensoriales/ultraestructuraRESUMEN
The limbic system-associated membrane protein (LAMP) is a glycosylphosphatidylinositol-anchored glycoprotein with three immunoglobulin (Ig) domains that can either enhance or inhibit neurite outgrowth depending upon the neuronal population examined. In the present study, we investigate the domains responsible for these activities. Domain deletion revealed that the N-terminal IgI domain is necessary and sufficient for the neurite-promoting activity observed in hippocampal neurons. In contrast, inhibition of neurite outgrowth in SCG neurons, which is mediated by heterophilic interactions, requires full-length LAMP, although selective inhibition of the second Ig domain, but not the first or third domains, prevented the inhibitory effect. This indicates that the IgII domain of LAMP harbors the neurite-inhibiting activity, but only in the context of the full-length configuration. Covasphere-binding analyses demonstrate IgI/IgI interactions, but no interaction between IgII and any other domain, consistent with the biological activities that each domain mediates. The data suggest that LAMP may serve as a bifunctional guidance molecule, with distinct structural domains contributing to the promotion and inhibition of neurite outgrowth.