Dissecting the Mechanisms of Hepcidin and BMP-SMAD Pathway Regulation By FKBP12

Abstract The BMP-SMAD pathway is activated when a dimeric ligand (BMP) interacts with a dimeric serine threonine kinase receptor (BMPRII) and triggers the activation of a dimeric BMP type I receptor (BMPRI). Catalytically active BMPRIs phosphorylate SMAD1/5/8 that, upon SMAD4 binding, translocate to the nucleus to regulate the expression of BMP target genes, including hepcidin. Hepcidin is the main regulator of iron homeostasis that controls body iron levels by binding and blocking the sole iron exporter ferroportin. In agreement, hepcidin expression is homeostatically activated by serum and liver iron, and its deficiency is a common hallmark of Hereditary Hemochromatosis (HH) and the major cause of iron overload in beta thalassemia. The components of the BMP-SMAD pathway relevant for hepcidin regulation are ALK2 and ALK3 (BMPRI); BMPR2 and ACVR2A (BMPRII), BMP2 and BMP6 (BMP ligands). Recently, we have identified the immunophilin FKBP12 as an inhibitor of hepcidin and demonstrated that FKBP12 binds ALK2 to avoid ligand-independent activation of the BMP-SMAD pathway. To investigate the mechanism of BMP-SMAD pathway and hepcidin regulation by FKBP12, we performed in vitro, ex vivo and in vivo studies. We found that FKBP12 sequestration by the immunosuppressive drug Tacrolimus (TAC) stabilizes ALK2-ALK2 homodimers and ALK2-ALK3 heterodimers in a transfected human hepatoma cell line. In addition, it increases the interaction of ALK2 with ACVR2A and BMPR2. To investigate the role of FKBP12 on BMP-SMAD signaling, BMPRI and II were silenced in murine primary hepatocytes. Despite FKBP12 co-immunoprecipitates only with ALK2, silencing of Alk2 and Alk3 completely blunts TAC-mediated BMP-SMAD pathway activation, suggesting that FKBP12 functionally interacts also with ALK3. Acvr2a silencing impairs TAC-dependent hepcidin upregulation, whereas Bmpr2 silencing does not. As expected, Fkbp12 silencing abrogates hepcidin upregulation by TAC, confirming the main role of this immunophilin in hepcidin regulation. In vivo, TAC treatment upregulates hepcidin in wild type and HH mouse models, but surprisingly, Fkbp12 mRNA downregulation by ASOs does not. Interestingly, Fkbp 2, 4 and 8 are highly expressed in murine hepatocytes and, according to literature data, are able to bind to TAC. Of note, Fkbp12 is the least expressed immunophilin in murine primary hepatocytes. To further investigate the FKBPs involved in TAC-dependent hepcidin regulation, Fkbp2, 4 and 8 were knockdown in murine primary HCs that were then treated with TAC. The TAC effect is preserved in siFkbp2- and siFkbp4-derived HCs, but abolished when Fkbp8 was downregulated. Overall these data suggest that: 1) FKBP12 regulates BMP-SMAD signaling by favoring ALK2-ALK3 homo and heterodimerization, and interaction with BMPRII in the absence of ligands; 2) TAC-mediated hepcidin upregulation is dependent upon ALK2, ALK3, ACVR2A, FKBP12 and FKBP8. 3) In vivo, TAC treatment upregulates hepcidin whereas Fkbp12 silencing does not, suggesting the existence of redundancy between the different FKBPs. Further studies are needed to dissect the role of FKBP8 in BMP-SMAD pathway and hepcidin regulation. Disclosures Aghajan: Ionis Pharmaceuticals, Inc.: Current Employment. Muckenthaler: Silence Therapeutics: Research Funding. Guo: Ionis Pharmaceuticals, Inc.: Current Employment.

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The transcriptional landscape of a hepatoma cell line grown on scaffolds of extracellular matrix proteins

BMC Genomics ◽

10.1186/s12864-021-07532-2 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Souvik Ghosh ◽

Anastasiya Börsch ◽

Shreemoyee Ghosh ◽

Mihaela Zavolan

Keyword(s):

Cell Culture ◽

Extracellular Matrix ◽

Drug Targets ◽

Hepatoma Cell ◽

Growth Potential ◽

Collagen I ◽

Matrix Proteins ◽

Hepatoma Cell Line ◽

Primary Hepatocytes

Abstract Background The behavior of cells in vivo is complex and highly dynamic, as it results from an interplay between intercellular matrix proteins with surface receptors and other microenvironmental cues. Although the effects of the cellular niche have been investigated for a number of cell types using different molecular approaches, comprehensive assessments of how the global transcriptome responds to 3D scaffolds composed of various extracellular matrix (ECM) constituents at different concentrations are still lacking. Results In this study, we explored the effects of two diverse extracellular matrix (ECM) components, Collagen I and Matrigel, on the transcriptional profile of cells in a cell culture system. Culturing Huh-7 cells on traditional cell culture plates (Control) or on the ECM components at different concentrations to modulate microenvironment properties, we have generated transcriptomics data that may be further explored to understand the differentiation and growth potential of this cell type for the development of 3D cultures. Our analysis infers transcription factors that are most responsible for the transcriptome response to the extracellular cues. Conclusion Our data indicates that the Collagen I substrate induces a robust transcriptional response in the Huh-7 cells, distinct from that induced by Matrigel. Enhanced hepatocyte markers (ALB and miR-122) reveal a potentially robust remodelling towards primary hepatocytes. Our results aid in defining the appropriate culture and transcription pathways while using hepatoma cell lines. As systems mimicking the in vivo structure and function of liver cells are still being developed, our study could potentially circumvent bottlenecks of limited availability of primary hepatocytes for preclinical studies of drug targets.

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Inhibiting the Immunophilin FKBP12: A Potential Therapeutic Approach to Iron Overload/Low Hepcidin Disorders

Blood ◽

10.1182/blood-2018-99-118112 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2333-2333

Author(s):

Mariateresa Pettinato ◽

Mariam Aghajan ◽

Alessandro Dulja ◽

Antonella Nai ◽

Violante Olivari ◽

...

Keyword(s):

Iron Overload ◽

Serum Iron ◽

Target Genes ◽

Hereditary Hemochromatosis ◽

Drug Repurposing ◽

Type I ◽

Smad Pathway ◽

Hepcidin Expression

Abstract Introduction Hepcidin negatively regulates body iron by binding and degrading the iron exporter ferroportin. Its expression is mainly controlled by the liver BMP-SMAD pathway whose activation requires BMP ligands (BMP2 and BMP6), constitutively active BMP type II receptors and the type I receptors ALK2 and ALK3. The co-receptor hemojuvelin (HJV) further potentiates the signaling. Mutations in key genes of the pathway impair hepcidin synthesis in Hereditary Hemochromatosis (HH), one of the most severe form being due to HJV mutations. As current therapies are symptomatic and do not correct hepcidin deficiency, alternative targeted therapies to increase hepcidin production are needed. We have demonstrated that the immunophilin FKBP12 binds ALK2 in hepatoma cells to prevent uncontrolled activation of the BMP pathway in the absence of ligands. We also demonstrated that FKBP12 sequestration by immunosuppressive drugs, such as FK506 (tacrolimus, TAC) or rapamycin, upregulate hepcidin in vitro. The role of FKBP12 is maintained in vivo since acute TAC treatment of wild type (WT) mice increases hepcidin expression (Colucci et al., Blood 2017). The aim of this study is to investigate whether pharmacologic inactivation of FKBP12, by TAC or antisense oligonucleotides (ASO), upregulates hepcidin for therapeutic purposes. Methods Primary hepatocytes isolated from WT, Hjv and Tfr2 KO mice (sv129/j background) were treated with increasing concentrations of TAC. Hepcidin and Id1 expression was investigated by qRT-PCR. Nine-weeks-old Hjv KO male mice were treated for 28 days with TAC (0.37 mg/h) delivered through surgically implanted mini-osmotic pumps. Six-weeks-old WT mice, were treated twice a week for 6 weeks with 50 mg/kg of Fkbp12 or control ASO. Mice were sacrificed and analyzed for iron, CBC, erythropoiesis and liver expression of hepcidin and BMP-SMAD target genes. Results TAC treatment of primary HCs from Hjv KO (Colucci et al., Blood 2018) and Tfr2 KO mice upregulates hepcidin as in WT mice, suggesting that HJV and TFR2 are dispensable for FKBP12-dependent hepcidin regulation and providing the proof of principle for FKBP12 targeting in HH. First, we explored a drug repurposing approach in the severe Hjv KO mice by chronic subcutaneous delivery of suboptimal, non-immunosuppressive TAC doses. Treatment of Hjv KO mice with TAC upregulates hepcidin via BMP-SMAD pathway activation, as assessed by Id1 and Smad7 upregulation. Since TAC also inhibits calcineurin upon FKBP12 sequestration and to avoid potential off-targets effect, FKBP12 was inactivated by ASO. Fkbp12 ASO treatment of WT mice decreases Fkbp12 expression by about 70-80% in liver, spleen and kidney, but not in the bone marrow. Fkbp12 ASO-treated mice exhibit microcytic anemia, decreased serum iron and upregulation of liver BMP-SMAD target genes. However, hepcidin remains inappropriately high considering the low serum iron. Fkbp12 ASO-treated mice show increased spleen immature erythroid precursors and increased expression of erythroferrone (Erfe). This is due to the unexpected effect of FKBP12 on spleen erythropoiesis and likely explains the lack of hepcidin upregulation. Conclusions Chronic TAC treatment in Hjv KO mice, which causes FKBP12 sequestration, improves hepcidin expression via BMP-SMAD pathway and favors spleen iron retention, suggesting that this "drug repurposing approach" may be beneficial for all iron overload disorders exhibiting low hepcidin. ASO-Fkbp12 in WT mice efficiently downregulates hepatic Fkbp12, upregulates the BMP-SMAD pathway but leaves hepcidin unchanged compared to control mice. We hypothesize that this result reflects the concomitant hepcidin inhibition due to decreased serum iron and increased Erfe expression. The latter is a consequence of spleen Fkbp12 reduction in ASO-treated mice, suggesting that a hepatocyte targeting Fkbp12 ASO is required for therapeutic purposes. Since Fkbp12 binds and inhibits ALK2 (Colucci et al., Blood 2017), our in vitro and in vivo data suggest that HJV and TFR2 functionally interact with ALK3 but not with ALK2. Disclosures Aghajan: Ionis Pharmaceuticals, Inc: Employment. Guo:Ionis Pharmaceuticals, Inc: Employment. Camaschella:vifor Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees.

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The Immunophilin FKBP12 Inhibits Hepcidin By Modulating BMP Type I-Type II Receptors Interaction and Ligand Responsiveness

Blood ◽

10.1182/blood-2019-130058 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 430-430

Author(s):

Alessandro Dulja ◽

Alessia Pagani ◽

Mariateresa Pettinato ◽

Antonella Nai ◽

Clara Camaschella ◽

...

Keyword(s):

Hepatoma Cell Line ◽

Type I ◽

Type Ii ◽

Smad Pathway ◽

Hepcidin Expression ◽

Receptor Complexes ◽

Bmp Receptors ◽

Pathway Activation ◽

Huh7 Cells

Introduction The liver hormone hepcidin is the master regulator of iron metabolism that modulates iron release into the circulation by binding and blocking the iron exporter ferroportin (Nemeth et al., 2004). Hepcidin expression is under the control of the BMP-SMAD pathway (Babitt et al., 2006), whose activation requires the formation of a hexameric complex composed of a dimer of BMP receptors type I (BMPR-Is), a dimer of BMPR type II (BMPR-IIs) and dimeric ligands. ALK2 and ALK3, as BMPR-Is (Steinbiecker et al., 2011), BMPR2 and ACVR2A, as BMPR-IIs (Mayeur et al., 2014), and BMP2 (Koch et al, 2017) and BMP6 (Meynard et al., 2009), as ligands, control hepcidin expression in vivo. We previously demonstrated that the immunophilin FKBP12 limits hepcidin expression in hepatocytes by binding ALK2 (Colucci et al., 2017). However, the molecular mechanism whereby FKBP12 regulates ALK2 and its relationship with BMPR-IIs and ligands in the regulation of the BMP-SMAD pathway and hepcidin expression are still unclear. Methods: BMPR-Is dimerization was evaluated by co-immunoprecipitation (CoIP) experiments performed in the HuH7 human hepatoma cell line. BMP-SMAD pathway and hepcidin promoter activation were analyzed by using a reporter vector with the luciferase under the control of BMP responsive elements or of the human hepcidin promoter, respectively. Endogenous hepcidin expression was measured by qRT-PCR. Results: Since BMPRIs act as dimers, we first tested whether FKBP12 modulates the dimerization process. MYC- and FLAG-tagged ALK2 or ALK3 were transfected in HuH7 cells in the presence of FKBP12. Cells were treated or not with tacrolimus (TAC), an immunosuppressive drug that sequesters FKBP12 from ALK2. FKBP12 promotes ALK2 homodimers, functionally inactive in the absence of ligands, with no effect on ALK3 homodimerization. TAC promotes increased ALK2 homodimerization and SMAD1/5/8 phosphorylation, demonstrating that in the absence of FKBP12, ALK2 homodimers are stabilized and functionally active. We next focused on BMP6, the physiologic ligand that binds preferentially ALK2 and plays a fundamental role in hepcidin regulation in vivo. In HuH7 cells transfected with FKBP12 and ALK2, BMP6 treatment reduced FKBP12-ALK2 binding and increased ALK2 homodimers. In agreement, SMAD1/5/8 phosphorylation was increased, indicating that FKBP12 displacement allows the formation of functional receptor complexes responsive to BMP6. BMPR-Is activate SMAD1/5/8 following BMPR-IIs phosphorylation. Since TAC induces SMAD1/5/8 phosphorylation in the absence of ligands, we hypothesized that FKBP12 displacement also affects the formation of BMPR-I/BMPR-II oligomers. HuH7 cells were transfected with ALK2, BMPR2 or ACVR2A and FKBP12, and treated or not with TAC. FKBP12 sequestration by TAC enhances the ALK2-BMPR2 and ALK2-ACVR2A interaction and accordingly activates SMAD1/5/8 signaling. Given that FKBP12 modulates BMP receptor interaction, we wondered how this functionally impacts on the response to BMP ligands, as BMP2, that guarantees basal hepcidin levels by binding ALK3, and BMP6, upregulated in iron overload that signals preferentially through ALK2. ALK3 upregulates the BMP pathway and hepcidin expression in a similar way in response to BMP2 and BMP6, in agreement with the evidence that both ligands bind ALK3. ALK2, which failed to activate the pathway in the absence ligands, leads to a greater response to BMP6, consistent with the fact that it is the BMP6 receptor. Thus FKBP12 quantitatively, rather than qualitatively, modulates the BMP-SMAD pathway activation in response to BMP ligands. Conclusions: Altogether our results clarify the molecular mechanisms of hepcidin regulation demonstrating that: 1) FKBP12 limits hepcidin expression by inducing the formation of inactive ALK2 homodimers in the absence of ligands. 2) Decreased FKBP12 binding to ALK2, by TAC or BMP6, favors the formation of active ALK2 homodimers. 3) FKBP12 sequestration increases the binding of ALK2 with the BMPR-IIs, thus favoring SMAD1/5/8 phosphorylation and pathway activation. 4) FKBP12 quantitatively modulates the response of BMPRIs to the ligands BMP2 and BMP6. Disclosures Camaschella: Vifor Iron Core: Consultancy; Celgene: Consultancy; Novartis: Consultancy.

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Overexpression of acyl-CoA synthetase-1 increases lipid deposition in hepatic (HepG2) cells and rodent liver in vivo

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00112.2006 ◽

2006 ◽

Vol 291 (4) ◽

pp. E737-E744 ◽

Cited By ~ 55

Author(s):

Heidi A. Parkes ◽

Elaine Preston ◽

Donna Wilks ◽

Mercedes Ballesteros ◽

Lee Carpenter ◽

...

Keyword(s):

Fatty Acid ◽

Hepg2 Cells ◽

Hepatoma Cell ◽

Acid Oxidation ◽

Hepatoma Cell Line ◽

Human Hepatoma Cell ◽

Chain Acyl

Accumulation of intracellular lipid in obesity is associated with metabolic disease in many tissues including liver. Storage of fatty acid as triglyceride (TG) requires the activation of fatty acids to long-chain acyl-CoAs (LC-CoA) by the enzyme acyl-CoA synthetase (ACSL). There are five known isoforms of ACSL (ACSL1, -3, -4, -5, -6), which vary in their tissue specificity and affinity for fatty acid substrates. To investigate the role of ACSL1 in the regulation of lipid metabolism, we used adenoviral-mediated gene transfer to overexpress ACSL1 in the human hepatoma cell-line HepG2 and in liver of rodents. Infection of HepG2 cells with the adenoviral construct AdACSL1 increased ACSL activity >10-fold compared with controls after 24 h. HepG2 cells overexpressing ACSL1 had a 40% higher triglyceride (TG) content (93 ± 3 vs. 67 ± 2 nmol/mg protein in controls, P < 0.05) after 24-h exposure to 1 mM oleate. Furthermore, ACSL1 overexpression produced a 60% increase in cellular LCA-CoA content (160 ± 6 vs. 100 ± 6 nmol/g protein in controls, P < 0.05) and increased [14C]oleate incorporation into TG without significantly altering fatty acid oxidation. In mice, AdACSL1 administration increased ACSL1 mRNA and protein more than fivefold over controls at 4 days postinfection. ACSL1 overexpression caused a twofold increase in TG content in mouse liver (39 ± 4 vs. 20 ± 2 μmol/g wet wt in controls, P < 0.05), and overexpression in rat liver increased [1-14C]palmitate clearance into liver TG. These in vitro and in vivo results suggest a pivotal role for ACSL1 in regulating TG synthesis in liver.

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Concurrent YAP/TAZ and SMAD signaling mediate vocal fold fibrosis

Scientific Reports ◽

10.1038/s41598-021-92871-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Ryosuke Nakamura ◽

Nao Hiwatashi ◽

Renjie Bing ◽

Carina P. Doyle ◽

Ryan C. Branski

Keyword(s):

Vocal Fold ◽

Vocal Folds ◽

Nuclear Accumulation ◽

Smad Pathway ◽

Surgical Interventions ◽

Smad Signaling ◽

Iatrogenic Damage ◽

Tgf Β1

AbstractVocal fold (VF) fibrosis is a major cause of intractable voice-related disability and reduced quality of life. Excision of fibrotic regions is suboptimal and associated with scar recurrence and/or further iatrogenic damage. Non-surgical interventions are limited, putatively related to limited insight regarding biochemical events underlying fibrosis, and downstream, the lack of therapeutic targets. YAP/TAZ integrates diverse cell signaling events and interacts with signaling pathways related to fibrosis, including the TGF-β/SMAD pathway. We investigated the expression of YAP/TAZ following vocal fold injury in vivo as well as the effects of TGF-β1 on YAP/TAZ activity in human vocal fold fibroblasts, fibroblast-myofibroblast transition, and TGF-β/SMAD signaling. Iatrogenic injury increased nuclear localization of YAP and TAZ in fibrotic rat vocal folds. In vitro, TGF-β1 activated YAP and TAZ in human VF fibroblasts, and inhibition of YAP/TAZ reversed TGF-β1-stimulated fibroplastic gene upregulation. Additionally, TGF-β1 induced localization of YAP and TAZ in close proximity to SMAD2/3, and nuclear accumulation of SMAD2/3 was inhibited by a YAP/TAZ inhibitor. Collectively, YAP and TAZ were synergistically activated with the TGF-β/SMAD pathway, and likely essential for the fibroplastic phenotypic shift in VF fibroblasts. Based on these data, YAP/TAZ may evolve as an attractive therapeutic target for VF fibrosis.

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Essential role of IPS-1 in innate immune responses against RNA viruses

Journal of Experimental Medicine ◽

10.1084/jem.20060792 ◽

2006 ◽

Vol 203 (7) ◽

pp. 1795-1803 ◽

Cited By ~ 345

Author(s):

Himanshu Kumar ◽

Taro Kawai ◽

Hiroki Kato ◽

Shintaro Sato ◽

Ken Takahashi ◽

...

Keyword(s):

Rna Viruses ◽

Rna Virus ◽

Critical Role ◽

Nuclear Factor Κb ◽

Type I ◽

Innate Immune Responses ◽

Antiviral Responses ◽

Deficient Mice

IFN-β promoter stimulator (IPS)-1 was recently identified as an adapter for retinoic acid–inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (Mda5), which recognize distinct RNA viruses. Here we show the critical role of IPS-1 in antiviral responses in vivo. IPS-1–deficient mice showed severe defects in both RIG-I– and Mda5-mediated induction of type I interferon and inflammatory cytokines and were susceptible to RNA virus infection. RNA virus–induced interferon regulatory factor-3 and nuclear factor κB activation was also impaired in IPS-1–deficient cells. IPS-1, however, was not essential for the responses to either DNA virus or double-stranded B-DNA. Thus, IPS-1 is the sole adapter in both RIG-I and Mda5 signaling that mediates effective responses against a variety of RNA viruses.

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Pyrrolizidine Alkaloids Induce Cell Death in Human HepaRG Cells in a Structure-Dependent Manner

International Journal of Molecular Sciences ◽

10.3390/ijms22010202 ◽

2020 ◽

Vol 22 (1) ◽

pp. 202

Author(s):

Josephin Glück ◽

Julia Waizenegger ◽

Albert Braeuning ◽

Stefanie Hessel-Pras

Keyword(s):

Cell Death ◽

Cell Viability ◽

Pyrrolizidine Alkaloids ◽

Defense Mechanism ◽

Hepatoma Cell ◽

Hepatoma Cell Line ◽

Dependent Manner ◽

Human Hepatoma Cell

Pyrrolizidine alkaloids (PAs) are a group of secondary metabolites produced in various plant species as a defense mechanism against herbivores. PAs consist of a necine base, which is esterified with one or two necine acids. Humans are exposed to PAs by consumption of contaminated food. PA intoxication in humans causes acute and chronic hepatotoxicity. It is considered that enzymatic PA toxification in hepatocytes is structure-dependent. In this study, we aimed to elucidate the induction of PA-induced cell death associated with apoptosis activation. Therefore, 22 structurally different PAs were analyzed concerning the disturbance of cell viability in the metabolically competent human hepatoma cell line HepaRG. The chosen PAs represent the main necine base structures and the different esterification types. Open-chained and cyclic heliotridine- and retronecine-type diesters induced strong cytotoxic effects, while treatment of HepaRG with monoesters did not affect cell viability. For more detailed investigation of apoptosis induction, comprising caspase activation and gene expression analysis, 14 PA representatives were selected. The proapoptotic effects were in line with the potency observed in cell viability studies. In vitro data point towards a strong structure–activity relationship whose effectiveness needs to be investigated in vivo and can then be the basis for a structure-associated risk assessment.

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Non-alcoholic fatty liver disease in mice with hepatocyte-specific deletion of mitochondrial fission factor

Diabetologia ◽

10.1007/s00125-021-05488-2 ◽

2021 ◽

Author(s):

Yukina Takeichi ◽

Takashi Miyazawa ◽

Shohei Sakamoto ◽

Yuki Hanada ◽

Lixiang Wang ◽

...

Keyword(s):

Er Stress ◽

Mitochondrial Dynamics ◽

Mitochondrial Fission ◽

Primary Hepatocytes ◽

Alcoholic Fatty Liver ◽

Expression Of Genes ◽

Specific Deletion

Abstract Aims/hypothesis Mitochondria are highly dynamic organelles continuously undergoing fission and fusion, referred to as mitochondrial dynamics, to adapt to nutritional demands. Evidence suggests that impaired mitochondrial dynamics leads to metabolic abnormalities such as non-alcoholic steatohepatitis (NASH) phenotypes. However, how mitochondrial dynamics are involved in the development of NASH is poorly understood. This study aimed to elucidate the role of mitochondrial fission factor (MFF) in the development of NASH. Methods We created mice with hepatocyte-specific deletion of MFF (MffLiKO). MffLiKO mice fed normal chow diet (NCD) or high-fat diet (HFD) were evaluated for metabolic variables and their livers were examined by histological analysis. To elucidate the mechanism of development of NASH, we examined the expression of genes related to endoplasmic reticulum (ER) stress and lipid metabolism, and the secretion of triacylglycerol (TG) using the liver and primary hepatocytes isolated from MffLiKO and control mice. Results MffLiKO mice showed aberrant mitochondrial morphologies with no obvious NASH phenotypes during NCD, while they developed full-blown NASH phenotypes in response to HFD. Expression of genes related to ER stress was markedly upregulated in the liver from MffLiKO mice. In addition, expression of genes related to hepatic TG secretion was downregulated, with reduced hepatic TG secretion in MffLiKO mice in vivo and in primary cultures of MFF-deficient hepatocytes in vitro. Furthermore, thapsigargin-induced ER stress suppressed TG secretion in primary hepatocytes isolated from control mice. Conclusions/interpretation We demonstrated that ablation of MFF in liver provoked ER stress and reduced hepatic TG secretion in vivo and in vitro. Moreover, MffLiKO mice were more susceptible to HFD-induced NASH phenotype than control mice, partly because of ER stress-induced apoptosis of hepatocytes and suppression of TG secretion from hepatocytes. This study provides evidence for the role of mitochondrial fission in the development of NASH. Graphical abstract

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WNT-5A regulates TGF-β-related activities in liver fibrosis

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00160.2016 ◽

2017 ◽

Vol 312 (3) ◽

pp. G219-G227 ◽

Cited By ~ 21

Author(s):

Leonie Beljaars ◽

Sara Daliri ◽

Christa Dijkhuizen ◽

Klaas Poelstra ◽

Reinoud Gosens

Keyword(s):

Liver Fibrosis ◽

Functional Role ◽

Collagen Type ◽

Collagen Type I ◽

Matrix Proteins ◽

Type I ◽

Human Livers

WNT-5A is a secreted growth factor that belongs to the noncanonical members of the Wingless-related MMTV-integration family. Previous studies pointed to a connection between WNT-5A and the fibrogenic factor TGF-β warranting further studies into the functional role of WNT-5A in liver fibrosis. Therefore, we studied WNT-5A expressions in mouse and human fibrotic livers and examined the relation between WNT-5A and various fibrosis-associated growth factors, cytokines, and extracellular matrix proteins. WNT-5A gene and protein expressions were significantly increased in fibrotic mouse and human livers compared with healthy livers. Regression or therapeutic intervention in mice resulted in decreased hepatic WNT-5A levels paralleled by lower collagen levels. Immunohistochemical analysis showed WNT-5A staining in fibrotic septa colocalizing with desmin staining indicating WNT-5A expression in myofibroblasts. In vitro studies confirmed WNT-5A expression in this cell type and showed that TGF-β significantly enhanced WNT-5A expression in contrast to PDGF-BB and proinflammatory cytokines IL-1β and TNF-α. Additionally, TGF-β induces the expression of the WNT receptors FZD2 and FZD8. After silencing of WNT-5A, reduced levels of collagen type I, vimentin, and fibronectin in TGF-β-stimulated myofibroblasts were measured compared with nonsilencing siRNA-treated controls. Interestingly, the antifibrotic cytokine IFNγ suppressed WNT-5A in vitro and in vivo. IFNγ-treated fibrotic mice showed significantly less WNT-5A expression compared with untreated fibrotic mice. In conclusion, WNT-5A paralleled collagen I levels in fibrotic mouse and human livers. WNT-5A expression in myofibroblasts is induced by the profibrotic factor TGF-β and plays an important role in TGF-β-induced regulation of fibrotic matrix proteins, whereas its expression can be reversed upon treatment, both in vitro and in vivo. NEW & NOTEWORTHY This study describes the localization and functional role of WNT-5A in human and mouse fibrotic livers. Hepatic WNT-5A expression parallels collagen type I expression. In vivo and in vitro, the myofibroblasts were identified as the key hepatic cells producing WNT-5A. WNT-5A is under control of TGF-β and its activities are primarily profibrotic.

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ADAR1 function affects HPV replication and is associated to recurrent human papillomavirus-induced dysplasia in HIV coinfected individuals

Scientific Reports ◽

10.1038/s41598-019-56422-x ◽

2019 ◽

Vol 9 (1) ◽

Author(s):

Maria Pujantell ◽

Roger Badia ◽

Iván Galván-Femenía ◽

Edurne Garcia-Vidal ◽

Rafael de Cid ◽

...

Keyword(s):

Human Papillomavirus ◽

Immune Activation ◽

Hpv Infection ◽

Innate Immune ◽

Type I ◽

Innate Immune Activation ◽

Enhanced Expression

AbstractInfection by human papillomavirus (HPV) alters the microenvironment of keratinocytes as a mechanism to evade the immune system. A-to-I editing by ADAR1 has been reported to regulate innate immunity in response to viral infections. Here, we evaluated the role of ADAR1 in HPV infection in vitro and in vivo. Innate immune activation was characterized in human keratinocyte cell lines constitutively infected or not with HPV. ADAR1 knockdown induced an innate immune response through enhanced expression of RIG-I-like receptors (RLR) signaling cascade, over-production of type-I IFNs and pro-inflammatory cytokines. ADAR1 knockdown enhanced expression of HPV proteins, a process dependent on innate immune function as no A-to-I editing could be identified in HPV transcripts. A genetic association study was performed in a cohort of HPV/HIV infected individuals followed for a median of 6 years (range 0.1–24). We identified the low frequency haplotype AACCAT significantly associated with recurrent HPV dysplasia, suggesting a role of ADAR1 in the outcome of HPV infection in HIV+ individuals. In summary, our results suggest that ADAR1-mediated innate immune activation may influence HPV disease outcome, therefore indicating that modification of innate immune effectors regulated by ADAR1 could be a therapeutic strategy against HPV infection.

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