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4.
J Mol Med (Berl) ; 93(10): 1085-1093, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26141517

RESUMO

UNLABELLED: The current study aims to identify the pro-fibrogenic role of Gremlin, an endogenous antagonist of bone morphogenetic proteins (BMPs) in chronic pancreatitis (CP). CP is a highly debilitating disease characterized by progressive pancreatic inflammation and fibrosis that ultimately leads to exocrine and endocrine dysfunction. While transforming growth factor (TGF)-ß is a known key pro-fibrogenic factor in CP, the TGF-ß superfamily member BMPs exert an anti-fibrogenic function in CP as reported by our group recently. To investigate how BMP signaling is regulated in CP by BMP antagonists, the mouse CP model induced by cerulein was used. During CP induction, TGF-ß1 messenger RNA (mRNA) increased 156-fold in 2 weeks, a BMP antagonist Gremlin 1 (Grem1) mRNA levels increased 145-fold at 3 weeks, and increases in Grem1 protein levels correlated with increases in collagen deposition. Increased Grem1 was also observed in human CP pancreata compared to normal. Grem1 knockout in Grem1 (+/-) mice revealed a 33.2 % reduction in pancreatic fibrosis in CP compared to wild-type littermates. In vitro in isolated pancreatic stellate cells, TGF-ß induced Grem1 expression. Addition of the recombinant mouse Grem1 protein blocked BMP2-induced Smad1/5 phosphorylation and abolished BMP2's suppression effects on TGF-ß-induced collagen expression. Evidences presented herein demonstrate that Grem1, induced by TGF-ß, is pro-fibrogenic by antagonizing BMP activity in CP. KEY MESSAGES: • Gremlin is upregulated in human chronic pancreatitis and a mouse CP model in vivo. • Deficiency of Grem1 in mice attenuates pancreatic fibrosis under CP induction in vivo. • TGF-ß induces Gremlin mRNA and protein expression in pancreatic stellate cells in vitro. • Gremlin blocks BMP2 signaling and function in pancreatic stellate cells in vitro. • This study discloses a pro-fibrogenic role of Gremlin by antagonizing BMP activity in chronic pancreatitis.


Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Pancreatite Crônica/metabolismo , Animais , Proteína Morfogenética Óssea 2/antagonistas & inibidores , Células Cultivadas , Ceruletídeo , Colágeno/metabolismo , Feminino , Fibrose , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Masculino , Camundongos Transgênicos , Pâncreas/metabolismo , Pâncreas/patologia , Células Estreladas do Pâncreas/metabolismo , Pancreatite Crônica/induzido quimicamente , Pancreatite Crônica/patologia , RNA Mensageiro/metabolismo , Fator de Crescimento Transformador beta1/genética
5.
PLoS One ; 9(2): e89114, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24586530

RESUMO

Bone morphogenetic proteins (BMPs) have an anti-fibrogenic function in the kidney, lung, and liver. However, their role in chronic pancreatitis (CP) is unknown. The aim of this study was to define the anti-fibrogenic role of BMP signaling in the pancreas in vivo under CP induction. Mice with a deletion of BMP type II receptor (BMPR2(+/-)) were used in this study in comparison with wild-type mice. CP was induced by repetitive cerulein injection intraperitoneally for 4 weeks, and the severity of CP was evaluated. Pancreatic stellate cells (PSCs) were isolated from the mice and treated with BMP2 and TGF-ß in vitro, and extracellular matrix protein (ECM) production was measured. Smad and mitogen-activated protein kinase (MAPK) signaling was also evaluated. BMPR2(+/-) mice revealed a greater pancreatic fibrosis, PSC activation and leukocyte infiltration after CP induction compared to wild-type mice (P<0.05). Under CP induction, phospho (p)Smad1/5/8 was elevated in wild-type mice and this effect was abolished in BMPR2(+/-) mice; pSmad2 and pp38(MAPK) were further enhanced in BMPR2(+/-) mice compared to wild-type mice (P<0.05). In vitro, BMP2 inhibited TGF-ß-induced ECM protein fibronectin production in wild-type PSCs; this effect was abolished in BMPR2(+/-) PSCs (P<0.05). In BMPR2(+/-) PSCs, pSmad1/5/8 level was barely detectable upon BMP2 stimulation, while pSmad2 level was further enhanced by TGF-ß stimulation, compared to wild-type PSCs (P<0.05). BMPR2/Smad1/5/8 signaling plays a protective role against cerulein-induced pancreatic fibrosis by inhibiting Smad2 and p38(MAPK) signaling pathways.


Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Fibrose/metabolismo , Pâncreas/metabolismo , Pancreatopatias/metabolismo , Transdução de Sinais/fisiologia , Animais , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Ceruletídeo , Fibrose/induzido quimicamente , Fibrose/patologia , Camundongos , Camundongos Knockout , Pâncreas/patologia , Pancreatopatias/induzido quimicamente , Pancreatopatias/patologia , Índice de Gravidade de Doença
6.
Am J Pathol ; 181(3): 804-17, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22841474

RESUMO

Epigenetic changes are associated with the regulation of transcription of key cell regulatory genes [micro RNAs (miRNAs)] during different types of liver injury. This study evaluated the role of methylation-associated miRNA, miR-34a, in alcoholic liver diseases. We identified that ethanol feeding for 4 weeks significantly up-regulated 0.8% of known miRNA compared with controls, including miR-34a. Treatment of normal human hepatocytes (N-Heps) and cholangiocytes [human intrahepatic biliary epithelial cells (HiBECs)] with ethanol and lipopolysaccharide induced a significant increase of miR-34a expression. Overexpression of miR-34a decreased ethanol-induced apoptosis in both N-Heps and HiBECs. In support of the concept that the 5'-promoter region of miR-34a was noted to be embedded within a CpG island, the expression level of miR-34a was significantly increased after demethylation treatment in N-Heps and HiBECs. By methylation-specific PCR, we confirmed that miR-34a activation is associated with ethanol-linked hypomethylation of the miR-34a promoter. A combination of bioinformatics, dual-luciferase reporter assay, mass spectrometry, and Western blot analysis revealed that caspase-2 and sirtuin 1 are the direct targets of miR-34a. Furthermore, modulation of miR-34a also altered expression of matrix metalloproteases 1 and 2, the mediators involved in hepatic remodeling during alcoholic liver fibrosis. These findings provide the basis for an exciting field in which the epigenomic microRNAs of hepatic cells may be manipulated with potential therapeutic benefits in human alcoholic liver diseases.


Assuntos
Epigênese Genética , Hepatopatias Alcoólicas/genética , MicroRNAs/genética , Animais , Sequência de Bases , Caspase 2/metabolismo , Linhagem Celular , Movimento Celular/genética , Sobrevivência Celular/genética , Transformação Celular Neoplásica , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA/genética , Modelos Animais de Doenças , Etanol , Fígado Gorduroso/genética , Fígado Gorduroso/patologia , Inativação Gênica , Humanos , Fígado/metabolismo , Fígado/patologia , Hepatopatias Alcoólicas/enzimologia , Hepatopatias Alcoólicas/patologia , Metaloproteinases da Matriz/genética , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Sirtuína 1/genética , Sirtuína 1/metabolismo , DNA Metiltransferase 3B
7.
Hepatology ; 55(1): 209-21, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21932404

RESUMO

UNLABELLED: Functional pluripotent characteristics have been observed in specific subpopulations of hepatic cells that express some of the known cholangiocyte markers. Although evidence indicates that specific cytokines, granulocyte macrophage colony-stimulating factors (GM-CSFs), and stem cell factors (SCFs) may be candidate treatments for liver injury, the role of these cytokines in intrahepatic biliary epithelium remodeling is unknown. Thus, our aim was to characterize the specific cytokines that regulate the remodeling potentials of cholangiocytes after 70% partial hepatectomy (PH). The expression of the cytokines and their downstream signaling molecules was studied in rats after 70% PH by immunoblotting and in small and large murine cholangiocyte cultures (SMCCs and LMCCs) by immunocytochemistry and real-time polymerase chain reaction (PCR). There was a significant, stable increase in SCF and GM-CSF levels until 7 days after PH. Real-time PCR analysis revealed significant increases of key remodeling molecules, such as S100 calcium-binding protein A4 (S100A4) and miR-181b, after SCF plus GM-CSF administration in SMCCs. SMCCs produced significant amounts of soluble and bound SCFs and GM-CSFs in response to transforming growth factor-beta (TGF-ß). When SMCCs were incubated with TGF-ß plus anti-SCF+GM-CSF antibodies, there was a significant decrease in S100A4 expression. Furthermore, treatment of SMCCs with SCF+GM-CSF significantly increased matrix metalloproteinases (MMP-2 and MMP-9) messenger RNA as well as miR-181b expression, along with a reduction of metalloproteinase inhibitor 3. Levels of MMP-2, MMP-9, and miR-181b were also up-regulated in rat liver and isolated cholangiocytes after PH. CONCLUSION: Our data suggest that altered expression of SCF+GM-CSF after PH can contribute to biliary remodeling (e.g., post-transplantation) by functional deregulation of the activity of key signaling intermediates involved in cell expansion and multipotent differentiation.


Assuntos
Fator Estimulador de Colônias de Granulócitos/genética , Regeneração Hepática/fisiologia , Fígado/fisiologia , Fator de Células-Tronco/genética , Animais , Ductos Biliares Intra-Hepáticos/citologia , Ductos Biliares Intra-Hepáticos/fisiologia , Divisão Celular/fisiologia , Linhagem Celular Transformada , Citocinas/fisiologia , Células Epiteliais/citologia , Fator Estimulador de Colônias de Granulócitos/metabolismo , Hepatectomia , Hepatócitos/citologia , Hepatócitos/fisiologia , Humanos , Fígado/citologia , Masculino , Metaloproteinase 3 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , MicroRNAs/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/fisiologia , Ratos , Ratos Endogâmicos F344 , Receptores de Fator Estimulador de Colônias/genética , Receptores de Fator Estimulador de Colônias/metabolismo , Transdução de Sinais/fisiologia , Fator de Células-Tronco/metabolismo , Fator de Crescimento Transformador beta/metabolismo
8.
J Cell Mol Med ; 16(1): 160-73, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21352471

RESUMO

MicroRNAs are endogenous small non-coding RNAs that regulate gene expression and cancer development. A rare population of hepatocellular cancer stem cells (HSCs) holds the extensive proliferative and self-renewal potential necessary to form a liver tumour. We postulated that specific transcriptional factors might regulate the expression of microRNAs and subsequently modulate the expression of gene products involved in phenotypic characteristics of HSCs. We evaluated the expression of microRNA in human HSCs by microarray profiling, and defined the target genes and functional effects of two groups of microRNA regulated by IL-6 and transcriptional factor Twist. A subset of highly chemoresistant and invasive HSCs was screened with aberrant expressions of cytokine IL-6 and Twist. We demonstrated that conserved let-7 and miR-181 family members were up-regulated in HSCs by global microarray-based microRNA profiling followed by validation with real-time polymerase chain reaction. Importantly, inhibition of let-7 increases the chemosensitivity of HSCs to sorafenib and doxorubicin whereas silencing of miR-181 led to a reduction in HSCs motility and invasion. Knocking down IL-6 and Twist in HSCs significantly reduced let-7 and miR-181 expression and subsequently inhibited chemoresistance and cell invasion. We showed that let-7 directly targets SOCS-1 and caspase-3, whereas miR-181 directly targets RASSF1A, TIMP3 as well as nemo-like kinase (NLK). In conclusion, alterations of IL-6- and Twist-regulated microRNA expression in HSCs play a part in tumour spreading and responsiveness to chemotherapy. Our results define a novel regulatory mechanism of let-7/miR-181s suggesting that let-7 and miR-181 may be molecular targets for eradication of hepatocellular malignancies.


Assuntos
Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , MicroRNAs/metabolismo , Células-Tronco Neoplásicas/fisiologia , Animais , Antineoplásicos/farmacologia , Benzenossulfonatos/farmacologia , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Análise por Conglomerados , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos SCID , MicroRNAs/genética , Transplante de Neoplasias , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Niacinamida/análogos & derivados , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Compostos de Fenilureia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/fisiologia , Piridinas/farmacologia , Sorafenibe , Proteína 1 Relacionada a Twist/genética , Proteína 1 Relacionada a Twist/metabolismo
9.
Hepatology ; 54(5): 1718-28, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21793031

RESUMO

UNLABELLED: Cholangiocarcinoma (CCA) is a biliary cancer arising from damaged bile ducts. Epithelial-mesenchymal transition (EMT) occurs as epithelial cells begin to resemble mesenchymal cells leading to increased invasion potential as the extracellular matrix (ECM) degrades. Histamine exerts its effects by way of four receptors (H1-H4 HRs). Clobenpropit, a potent H4HR agonist, inhibits mammary adenocarcinoma growth. We have shown that (1) cholangiocytes and CCA cells express H1-H4 HRs and (2) the H3HR decreases CCA proliferation. We evaluated the effects of clobenpropit on CCA proliferation, invasion, EMT phenotypes, and ECM degradation. In vitro, we used CCA cell lines to study proliferation, signaling pathways, and the morphological invasive potential. Gene and protein expression of the hepatobiliary epithelial markers CK-7, CK-8, and CK-19, the focal contact protein paxillin, and the mesenchymal markers fibronectin, s100A4, and vimentin were evaluated. Cell invasion across an ECM layer was quantitated and matrix metalloproteinase-1, -2, -3, -9, and -11 gene and protein expression was examined. Evaluation of the specific role of H4HR was performed by genetic knockdown of the H3HR and overexpression of H4HR. Proliferation was evaluated by proliferating cellular nuclear antigen immunoblotting. In vivo, xenograft tumors were treated with either vehicle or clobenpropit for 39 days. Tumor volume was recorded every other day. Clobenpropit significantly decreased CCA proliferation by way of a Ca(2+) -dependent pathway and altered morphological development and invasion. Loss of H3HR expression or overexpression of H4HR significantly decreased CCA proliferation. In vivo, clobenpropit inhibited xenograft tumor growth compared with controls. CONCLUSION: Modulation of H4HR by clobenpropit disrupts EMT processes, ECM breakdown, and invasion potential and decreases tumor growth. Interruption of tumorigenesis and invasion by histamine may add to therapeutic advances for CCAs.


Assuntos
Neoplasias dos Ductos Biliares/tratamento farmacológico , Ductos Biliares Intra-Hepáticos/patologia , Colangiocarcinoma/tratamento farmacológico , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Imidazóis/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Tioureia/análogos & derivados , Animais , Apoptose/efeitos dos fármacos , Neoplasias dos Ductos Biliares/patologia , Biópsia , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Colangiocarcinoma/secundário , AMP Cíclico/metabolismo , Progressão da Doença , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/enzimologia , Antagonistas dos Receptores Histamínicos H3/farmacologia , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Camundongos , RNA Interferente Pequeno/farmacologia , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Histamínicos/genética , Receptores Histamínicos/metabolismo , Receptores Histamínicos H4 , Transdução de Sinais/efeitos dos fármacos , Tioureia/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
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