scholarly journals Metabolic Alterations in Spheroid-Cultured Hepatic Stellate Cells

2020 ◽  
Vol 21 (10) ◽  
pp. 3451 ◽  
Author(s):  
Koichi Fujisawa ◽  
Taro Takami ◽  
Nanami Sasai ◽  
Toshihiko Matsumoto ◽  
Naoki Yamamoto ◽  
...  
Keyword(s):  
Gene Expression ◽  
Hepatic Stellate Cells ◽  
Nuclear Translocation ◽  
Smooth Muscle Actin ◽  
Hippo Pathway ◽  
Lipid Synthesis ◽  
Stellate Cells ◽  
Vital Role ◽  
Metabolome Analysis ◽  
The Hippo Pathway

Hepatic stellate cells (HSCs) play a vital role in liver fibrosis, and a greater understanding of their regulation is required. Recent studies have focused on relationships between extracellular matrix (ECM) stiffness and gene expression or cellular metabolism, but none have provided a detailed metabolic analysis of HSC changes in spheroid cultures. Accordingly, in the present study, we created an HSC spheroid culture and analyzed changes in gene expression and metabolism. Expression of α-smooth muscle actin (α-SMA) decreased in the spheroids, suppressing proliferation. Gene expression analysis revealed the cell cycle, sirtuin signaling, mitochondrial dysfunction, and the Hippo pathway to be canonical pathways, believed to result from decreased proliferative ability or mitochondrial suppression. In the Hippo pathway, nuclear translocation of the yes-associated protein (YAP) was decreased in the spheroid, which was associated with the stiffness of the ECM. Metabolome analysis showed glucose metabolism changes in the spheroid, including glutathione pathway upregulation and increased lipid synthesis. Addition of the glycolytic product phosphoenolpyruvate (PEP) led to increased spheroid size, with increased expression of proteins such as α-SMA and S6 ribosomal protein (RPS6) phosphorylation, which was attributed to decreased suppression of translation. The results of our study contribute to the understanding of metabolic changes in HSCs and the progression of hepatic fibrosis.

scholarly journals Albumin inhibits the activation of hepatic stellate cells by suppressing TGF-β/Smad3 signaling via IL-1β

2019 ◽  
Author(s):  
Ji Hoon Park ◽  
Janghyun Kim ◽  
So-Young Choi ◽  
Kiweon Cha ◽  
Heekyung Park ◽  
...  
Keyword(s):  
Hepatic Stellate Cells ◽  
Target Genes ◽  
Nuclear Translocation ◽  
Smooth Muscle Actin ◽  
Stellate Cells ◽  
Collagen Type I ◽  
Retinol Binding Protein ◽  
Type I ◽  
Dependent Manner ◽  
Promising Therapeutic Approach

AbstractActivated hepatic stellate cells (HSCs) play a key role in liver fibrosis and inactivating HSCs has been considered a promising therapeutic approach. We previously showed that albumin and its derivative, retinol binding protein (RBP)-albumin domain III fusion protein (named R-III), inhibit HSC activation. Here, we investigate the mode of action of albumin and R-III. NF-κB in activated HSCs was evenly distributed in the cytoplasm, but albumin expression and R-III treatment (albumin/R-III) induced NF-κB nuclear translocation via retinoic acid (RA) sequestration, resulting in increased expression of interleukin-1β (IL-1β). In an IL-1β dependent manner, albumin/R-III inhibited Smad3 nuclear translocation via TAK1-, JNK-mediated Smad3 linker phosphorylation and decreased expression of Smad3 target genes, such as α-smooth muscle actin and collagen type I. Mutation of the Smad3 linker phosphorylation sites abolished R-III effects on Smad3. In conclusion, our data suggest that the anti-fibrotic effects of albumin/R-III are due to RA sequestration which downregulates RAR-mediated signaling and also TGF-β/Smad3 signaling. This mechanistic elucidation of albumin function in HSCs provides clues to understanding the frequent albumin mutations found in hepatocellular carcinoma.

scholarly journals Targeting IRF3 as a YAP agonist therapy against gastric cancer

2018 ◽  
Vol 215 (2) ◽  
pp. 699-718 ◽  
Author(s):  
Shi Jiao ◽  
Jingmin Guan ◽  
Min Chen ◽  
Wenjia Wang ◽  
Chuanchuan Li ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Target Genes ◽  
Nuclear Translocation ◽  
Patient Survival ◽  
Hippo Pathway ◽  
Vital Role ◽  
Dependent Manner ◽  
Genome Wide ◽  
The Hippo Pathway ◽  
Innate Antiviral Immunity

The Hippo pathway plays a vital role in tissue homeostasis and tumorigenesis. The transcription factor IRF3 is essential for innate antiviral immunity. In this study, we discovered IRF3 as an agonist of Yes-associated protein (YAP). The expression of IRF3 is positively correlated with that of YAP and its target genes in gastric cancer; the expression of both IRF3 and YAP is up-regulated and prognosticates patient survival. IRF3 interacts with both YAP and TEAD4 in the nucleus to enhance their interaction, promoting nuclear translocation and activation of YAP. IRF3 and YAP–TEAD4 are associated genome-wide to cobind and coregulate many target genes of the Hippo pathway. Overexpression of active IRF3 increased, but depletion of IRF3 reduced, the occupancy of YAP on the target genes. Knockdown or pharmacological targeting of IRF3 by Amlexanox, a drug used clinically for antiinflammatory treatment, inhibits gastric tumor growth in a YAP-dependent manner. Collectively, our study identifies IRF3 as a positive regulator for YAP, highlighting a new therapeutic target against YAP-driven cancers.

Norepinephrine induces calcium spikes and proinflammatory actions in human hepatic stellate cells

2006 ◽  
Vol 291 (5) ◽  
pp. G877-G884 ◽  
Author(s):  
Pau Sancho-Bru ◽  
Ramón Bataller ◽  
Jordi Colmenero ◽  
Xavier Gasull ◽  
Montserrat Moreno ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Portal Hypertension ◽  
Liver Fibrosis ◽  
Hepatic Stellate Cells ◽  
Molecular Mechanisms ◽  
Nuclear Translocation ◽  
Stellate Cells ◽  
Luciferase Reporter ◽  
Dependent Manner ◽  
Cell Contraction

Catecholamines participate in the pathogenesis of portal hypertension and liver fibrosis through α1-adrenoceptors. However, the underlying cellular and molecular mechanisms are largely unknown. Here, we investigated the effects of norepinephrine (NE) on human hepatic stellate cells (HSC), which exert vasoactive, inflammatory, and fibrogenic actions in the injured liver. Adrenoceptor expression was assessed in human HSC by RT-PCR and immunocytochemistry. Intracellular Ca2+ concentration ([Ca2+]i) was studied in fura-2-loaded cells. Cell contraction was studied by assessing wrinkle formation and myosin light chain II (MLC II) phosphorylation. Cell proliferation and collagen-α1(I) expression were assessed by [3H]thymidine incorporation and quantitative PCR, respectively. NF-κB activation was assessed by luciferase reporter gene and p65 nuclear translocation. Chemokine secretion was assessed by ELISA. Normal human livers expressed α1A-adrenoceptors, which were markedly upregulated in livers with advanced fibrosis. Activated human HSC expressed α1A-adrenoceptors. NE induced multiple rapid [Ca2+]i oscillations (Ca2+ spikes). Prazosin (α1-blocker) completely prevented NE-induced Ca2+ spikes, whereas propranolol (nonspecific β-blocker) partially attenuated this effect. NE caused phosphorylation of MLC II and cell contraction. In contrast, NE did not affect cell proliferation or collagen-α1(I) expression. Importantly, NE stimulated the secretion of inflammatory chemokines (RANTES and interleukin-8) in a dose-dependent manner. Prazosin blocked NE-induced chemokine secretion. NE stimulated NF-κB activation. BAY 11-7082, a specific NF-κB inhibitor, blocked NE-induced chemokine secretion. We conclude that NE stimulates NF-κB and induces cell contraction and proinflammatory effects in human HSC. Catecholamines may participate in the pathogenesis of portal hypertension and liver fibrosis by targeting HSC.

scholarly journals Inflammasome-mediated regulation of hepatic stellate cells

2009 ◽  
Vol 296 (6) ◽  
pp. G1248-G1257 ◽  
Author(s):  
Azuma Watanabe ◽  
Muhammad Adnan Sohail ◽  
Dawidson Assis Gomes ◽  
Ardeshir Hashmi ◽  
Jun Nagata ◽  
...  
Keyword(s):  
Liver Fibrosis ◽  
Immune Cells ◽  
Hepatic Stellate Cells ◽  
Smooth Muscle Actin ◽  
Stellate Cells ◽  
Inflammasome Activation ◽  
Cellular Injury ◽  
Wild Type ◽  
Primary Mouse ◽  
Msu Crystals

The inflammasome is a cytoplasmic multiprotein complex that has recently been identified in immune cells as an important sensor of signals released by cellular injury and death. Analogous to immune cells, hepatic stellate cells (HSC) also respond to cellular injury and death. Our aim was to establish whether inflammasome components were present in HSC and could regulate HSC functionality. Monosodium urate (MSU) crystals (100 μg/ml) were used to experimentally induce inflammasome activation in LX-2 and primary mouse HSC. Twenty-four hours later primary mouse HSC were stained with α-smooth muscle actin and visualized by confocal microscopy, and TGF-β and collagen1 mRNA expression was quantified. LX-2 cells were further cultured with or without MSU crystals for 24 h in a transwell chemotaxis assay with PDGF as the chemoattractant. We also examined inhibition of calcium (Ca2+) signaling in LX-2 cells treated with or without MSU crystals using caged inositol 1,4,5-triphosphate (IP3). Finally, we confirmed an important role of the inflammasome in experimental liver fibrosis by the injection of carbon tetrachloride (CCl4) or thioacetamide (TAA) in wild-type mice and mice lacking components of the inflammasome. Components of the inflammasome are expressed in LX-2 cells and primary HSC. MSU crystals induced upregulation of TGF-β and collagen1 mRNA and actin reorganization in HSCs from wild-type mice but not mice lacking inflammasome components. MSU crystals inhibited the release of Ca2+ via IP3 in LX-2 cells and also inhibited PDGF-induced chemotaxis. Mice lacking the inflammasome-sensing and adaptor molecules, NLRP3 and apoptosis-associated speck-like protein containing CARD, had reduced CCl4 and TAA-induced liver fibrosis. We concluded that inflammasome components are present in HSC, can regulate a variety of HSC functions, and are required for the development of liver fibrosis.

scholarly journals NPHP4, a cilia-associated protein, negatively regulates the Hippo pathway

2011 ◽  
Vol 193 (4) ◽  
pp. 633-642 ◽  
Author(s):  
Sandra Habbig ◽  
Malte P. Bartram ◽  
Roman U. Müller ◽  
Ricarda Schwarz ◽  
Nikolaos Andriopoulos ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cellular Proliferation ◽  
Nuclear Translocation ◽  
Hippo Pathway ◽  
Negative Regulator ◽  
Hippo Signaling ◽  
Transcriptional Coactivator ◽  
Hippo Signaling Pathway ◽  
The Hippo Pathway ◽  
Potent Negative Regulator

The conserved Hippo signaling pathway regulates organ size in Drosophila melanogaster and mammals and has an essential role in tumor suppression and the control of cell proliferation. Recent studies identified activators of Hippo signaling, but antagonists of the pathway have remained largely elusive. In this paper, we show that NPHP4, a known cilia-associated protein that is mutated in the severe degenerative renal disease nephronophthisis, acts as a potent negative regulator of mammalian Hippo signaling. NPHP4 directly interacted with the kinase Lats1 and inhibited Lats1-mediated phosphorylation of the Yes-associated protein (YAP) and TAZ (transcriptional coactivator with PDZ-binding domain), leading to derepression of these protooncogenic transcriptional regulators. Moreover, NPHP4 induced release from 14-3-3 binding and nuclear translocation of YAP and TAZ, promoting TEA domain (TEAD)/TAZ/YAP-dependent transcriptional activity. Consistent with these data, knockdown of NPHP4 negatively affected cellular proliferation and TEAD/TAZ activity, essentially phenocopying loss of TAZ function. These data identify NPHP4 as a negative regulator of the Hippo pathway and suggest that NPHP4 regulates cell proliferation through its effects on Hippo signaling.

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