scholarly journals PNPLA3 downregulation exacerbates the fibrotic response in human hepatic stellate cells

PLoS ONE ◽  
2021 ◽  
Vol 16 (12) ◽  
pp. e0260721
Author(s):  
Brian Rady ◽  
Takahiro Nishio ◽  
Debanjan Dhar ◽  
Xiao Liu ◽  
Mark Erion ◽  
...  
Keyword(s):  
Gene Expression ◽  
Hepatic Stellate Cells ◽  
Missense Variant ◽  
Stellate Cells ◽  
Mutant Form ◽  
Wild Type ◽  
Fibrotic Response ◽  
Human Hscs ◽  
Complications And Mortality

Non-alcoholic steatohepatitis (NASH) results, in part, from the interaction of metabolic derangements with predisposing genetic variants, leading to liver-related complications and mortality. The strongest genetic determinant is a highly prevalent missense variant in patatin-like phospholipase domain-containing protein 3 (PNPLA3 p.I148M). In human liver hepatocytes PNPLA3 localizes to the surface of lipid droplets where the mutant form is believed to enhance lipid accumulation and release of pro-inflammatory cytokines. Less is known about the role of PNPLA3 in hepatic stellate cells (HSCs). Here we characterized HSC obtained from patients carrying the wild type (n = 8 C/C) and the heterozygous (n = 6, C/G) or homozygous (n = 6, G/G) PNPLA3 I148M and investigated the effect of genotype and PNPLA3 downregulation on baseline and TGF-β-stimulated fibrotic gene expression. HSCs from all genotypes showed comparable baseline levels of PNPLA3 and expression of the fibrotic genes α-SMA, COL1A1, TIMP1 and SMAD7. Treatment with TGF-β increased PNPLA3 expression in all 3 genotypes (~2-fold) and resulted in similar stimulation of the expression of several fibrogenic genes. In primary human HSCs carrying wild-type (WT) PNPLA3, siRNA treatment reduced PNPLA3 mRNA by 79% resulting in increased expression of α-SMA, Col1a1, TIMP1, and SMAD7 in cells stimulated with TGF-β. Similarly, knock-down of PNPLA3 in HSCs carrying either C/G or G/G genotypes resulted in potentiation of TGF-β induced expression of fibrotic genes. Knockdown of PNPLA3 did not impact fibrotic gene expression in the absence of TGF-β treatment. Together, these data indicate that the presence of the I148M PNPLA3 mutation in HSC has no effect on baseline activation and that downregulation of PNPLA3 exacerbates the fibrotic response irrespective of the genotype.

scholarly journals Inhibition of SIRT2 suppresses hepatic fibrosis

2016 ◽  
Vol 310 (11) ◽  
pp. G1155-G1168 ◽  
Author(s):  
Maribel Arteaga ◽  
Na Shang ◽  
Xianzhong Ding ◽  
Sherri Yong ◽  
Scott J. Cotler ◽  
...  
Keyword(s):  
Gene Expression ◽  
Liver Fibrosis ◽  
Hepatic Fibrosis ◽  
Hepatic Stellate Cells ◽  
Histone Deacetylases ◽  
Critical Role ◽  
Stellate Cells ◽  
Novel Strategy ◽  
Underlying Mechanisms

Liver fibrosis can progress to cirrhosis and result in serious complications of liver disease. The pathogenesis of liver fibrosis involves the activation of hepatic stellate cells (HSCs), the underlying mechanisms of which are not fully known. Emerging evidence suggests that the classic histone deacetylases play a role in liver fibrosis, but the role of another subfamily of histone deacetylases, the sirtuins, in the development of hepatic fibrosis remains unknown. In this study, we found that blocking the activity of sirtuin 2 (SIRT2) by using inhibitors or shRNAs significantly suppressed fibrogenic gene expression in HSCs. We further demonstrated that inhibition of SIRT2 results in the degradation of c-MYC, which is important for HSC activation. In addition, we discovered that inhibition of SIRT2 suppresses the phosphorylation of ERK, which is critical for the stabilization of c-MYC. Moreover, we found that Sirt2 deficiency attenuates the hepatic fibrosis induced by carbon tetrachloride (CCl4) and thioacetamide (TAA). Furthermore, we showed that SIRT2, p-ERK, and c-MYC proteins are all overexpressed in human hepatic fibrotic tissues. These data suggest a critical role for the SIRT2/ERK/c-MYC axis in promoting hepatic fibrogenesis. Inhibition of the SIRT2/ERK/c-MYC axis represents a novel strategy to prevent and to potentially treat liver fibrosis and cirrhosis.

scholarly journals Determination of the Underlying Mechanisms for the Anti-fibrogenic Effect of n-3 Polyunsaturated Fatty Acids in Hepatic Stellate Cells (P08-105-19)

2019 ◽  
Vol 3 (Supplement_1) ◽  
Author(s):  
Siqi Hu ◽  
Minkyung Bae ◽  
Young-Ki Park ◽  
Ji-Young Lee
Keyword(s):  
Gene Expression ◽  
Fatty Acids ◽  
Fatty Acid ◽  
Hepatic Stellate Cells ◽  
Stellate Cells ◽  
Inhibitory Effect ◽  
Basal Expression ◽  
Epa And Dha ◽  
Human Hscs ◽  
Underlying Mechanisms

Abstract Objectives We previously reported that n-3 polyunsaturated fatty acids (PUFAs) exhibit potent anti-fibrogenic effects independent of the SMA- and MAD-related protein (SMAD3) pathway in primary human hepatic stellate cells (HSCs), the major cell type responsible for extracellular matrix production for the development of liver fibrosis. The objective of this study was to further elucidate the underlying mechanisms for the anti-fibrogenic effect of n-3 PUFAs in HSCs with a primary focus on peroxisome proliferator-activated receptor γ (PPARγ) as it is known to inhibit HSC activation. Methods n-3 PUFAs, including alpha linolenic acid (ALA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), were complexed with bovine serum albumin (BSA) at a BSA to fatty acid molar ratio of 1:2.5. Primary human HSCs were treated either with BSA vehicle control or 50 μM of a fatty acid for 24 h, after which they were activated with transforming growth factor β1 (TGFβ1; 4 ng/ml), a potent fibrogenic cytokine, for additional 24 h. Also, in another experiment, primary human HSCs were pre-treated with 10 μM of GW9662, a PPARγ antagonist, 18 h prior to the fatty acid and TGFβ1 treatments. Results ALA, EPA and DHA significantly decreased TGFβ1-induced mRNA expression of fibrogenic genes such as α-smooth muscle actin and collagen type I alpha 1 chain with EPA and DHA being more potent than ALA. The similar trend was also observed in their protein levels. In the absence of TGFβ1 treatment, EPA and DHA markedly increased PPARγ mRNA abundance. TGFβ1 significantly decreased PPARγ mRNA, which was attenuated by EPA and DHA, but not by ALA. Inhibition of PPARγ by GW9662 did not alter either the basal expression of PPARγ or the basal expression of fibrogenic genes. However, the inhibitory effect of EPA and DHA on the induction of fibrogenic gene expression by TGFβ1 was not diminished by GW9662. Conclusions n-3 PUFAs showed a potent inhibitory effect on TGFβ1-induced fibrogenic gene expression in primary human HSCs. Inhibition of PPARγ activity did not alter the anti-fibrogenic effect of n-3 PUFAs. The results suggest that n-3 PUFAs, particularly EPA and DHA to a similar degree, exert their anti-fibrogenic effect in primary human HSCs independent of the activation of PPARγ pathway. Funding Sources This study was supported by USDA Multistate Hatch and USDA Hatch.

scholarly journals The nucleoplasmic interactions among Lamin A/C-pRB-LAP2α-E2F1 are modulated by dexamethasone

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Anastasia Ricci ◽  
Sara Orazi ◽  
Federica Biancucci ◽  
Mauro Magnani ◽  
Michele Menotta
Keyword(s):  
Gene Expression ◽  
Cell Cycle Progression ◽  
Regulation Of Gene Expression ◽  
Lamin A ◽  
Wild Type ◽  
Cycle Progression ◽  
C Dynamics ◽  
E2f Transcription Factor ◽  
Transcription Factor 1

AbstractAtaxia telangiectasia (AT) is a rare genetic neurodegenerative disease. To date, there is no available cure for the illness, but the use of glucocorticoids has been shown to alleviate the neurological symptoms associated with AT. While studying the effects of dexamethasone (dex) in AT fibroblasts, by chance we observed that the nucleoplasmic Lamin A/C was affected by the drug. In addition to the structural roles of A-type lamins, Lamin A/C has been shown to play a role in the regulation of gene expression and cell cycle progression, and alterations in the LMNA gene is cause of human diseases called laminopathies. Dex was found to improve the nucleoplasmic accumulation of soluble Lamin A/C and was capable of managing the large chromatin Lamin A/C scaffolds contained complex, thus regulating epigenetics in treated cells. In addition, dex modified the interactions of Lamin A/C with its direct partners lamin associated polypeptide (LAP) 2a, Retinoblastoma 1 (pRB) and E2F Transcription Factor 1 (E2F1), regulating local gene expression dependent on E2F1. These effects were differentially observed in both AT and wild type (WT) cells. To our knowledge, this is the first reported evidence of the role of dex in Lamin A/C dynamics in AT cells, and may represent a new area of research regarding the effects of glucocorticoids on AT. Moreover, future investigations could also be extended to healthy subjects or to other pathologies such as laminopathies since glucocorticoids may have other important effects in these contexts as well.

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.

Retrovirus-induced interference with collagen I gene expression in Mov13 fibroblasts is maintained in the absence of DNA methylation

1991 ◽  
Vol 11 (1) ◽  
pp. 47-54
Author(s):  
H Chan ◽  
S Hartung ◽  
M Breindl
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Chromatin Structure ◽  
Transcriptional Activity ◽  
Xenopus Laevis Oocytes ◽  
Regulatory Sequences ◽  
Type I ◽  
Wild Type ◽  
Col1a1 Gene

We have studied the role of DNA methylation in repression of the murine alpha 1 type I collagen (COL1A1) gene in Mov13 fibroblasts. In Mov13 mice, a retroviral provirus has inserted into the first intron of the COL1A1 gene and blocks its expression at the level of transcriptional initiation. We found that regulatory sequences in the COL1A1 promoter region that are involved in the tissue-specific regulation of the gene are unmethylated in collagen-expressing wild-type fibroblasts and methylated in Mov13 fibroblasts, confirming and extending earlier observations. To directly assess the role of DNA methylation in the repression of COL1A1 gene transcription, we treated Mov13 fibroblasts with the demethylating agent 5-azacytidine. This treatment resulted in a demethylation of the COL1A1 regulatory sequences but failed to activate transcription of the COL1A1 gene. Moreover, the 5-azacytidine treatment induced a transcription-competent chromatin structure in the retroviral sequences but not in the COL1A1 promoter. In DNA transfection and microinjection experiments, we found that the provirus interfered with transcriptional activity of the COL1A1 promoter in Mov13 fibroblasts but not in Xenopus laevis oocytes. In contrast, the wild-type COL1A1 promoter was transcriptionally active in Mov13 fibroblasts. These experiments showed that the COL1A1 promoter is potentially transcriptionally active in the presence of proviral sequences and that Mov13 fibroblasts contain the trans-acting factors required for efficient COL1A1 gene expression. Our results indicate that the provirus insertion in Mov13 can inactivate COL1A1 gene expression at several levels. It prevents the developmentally regulated establishment of a transcription-competent methylation pattern and chromatin structure of the COL1A1 domain and, in the absence of DNA methylation, appears to suppress the COL1A1 promoter in a cell-specific manner, presumably by assuming a dominant chromatin structure that may be incompatible with transcriptional activity of flanking cellular sequences.

scholarly journals Effect of Fibroblast Growth Factor 21 on the Expression of TLR4 and BAMBI Genes in Cholesterol-Treated Human Hepatic Stellate Cells

2021 ◽  
Vol 12 (3) ◽  
Author(s):  
Elham Shakerian ◽  
Narges Mohammad Taghvaei ◽  
Zohre Askari ◽  
Reza Afarin
Keyword(s):  
Growth Factor ◽  
Mrna Expression ◽  
Fibroblast Growth Factor ◽  
Hepatic Stellate Cells ◽  
Stellate Cells ◽  
Fibroblast Growth Factor 21 ◽  
Control Group ◽  
Type I ◽  
Fibroblast Growth ◽  
Human Hscs

Background: Activated hepatic stellate cells (HSCs) are the primary mediators in the progression of hepatic fibrosis. The activation of toll-like receptor 4 (TLR4) signaling leads to the downregulation of the transmembrane inhibitory transforming growth factor-beta (TGF-β) pseudoreceptor BMP and activin membrane-bound inhibitor (BAMBI) on HSCs. Fibroblast growth factor 21 (FGF21) is a natural secretory protein in the body with effects, such as the reduction of fat accumulation and oxidation of lipids; however; no study has investigated FGF21 ability to prevent the progression of liver fibrosis. Objectives: This study aimed to examine the beneficial effects of FGF21 to reduce cholesterol-activated human HSCs. Methods: The human HSCs were incubated in media containing different concentrations of cholesterol, including 25, 50, 75, 100, 125, and 150 μM, for 24 h and then incubated with FGF21 for 24 h. Total ribonucleic acids were extracted and reversely transcribed into complementary deoxyribonucleic acid. A quantitative real-time polymerase chain reaction was performed in this study. Results: The results showed that the messenger ribonucleic acid (mRNA) expression of TGF-β, collagen, type I, alpha 1 (collagen1α), and TLR4 genes increased significantly in the presence of cholesterol (75 and 100 μM), compared to that of the control group (* P < 0.05, ** P < 0.01, and *** P < 0.001); nevertheless, the mRNA expression of the BAMBI gene significantly reduced, compared to that of the control group (* P < 0.05). The FGF21 significantly reduced the mRNA expression of TGF-β, collagen1α, and TLR4 genes (# P < 0.05). The mRNA expression of the BAMBI gene significantly increased with FGF21 (# P < 0.05). Conclusions: It was concluded that the treatment with FGF21 reduces the cholesterol-activated HSCs by decreasing the mRNA expression of the TLR4, TGF-β, and collagen1α genes and increasing the mRNA expression of the BAMBI gene.

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