Hepatic prohibitin 1 and methionine adenosyltransferase α1 defend against primary and secondary liver cancer metastasis.

BACKGROUND & AIMS: The liver is a common site of cancer metastasis, most commonly from colorectal cancer, and primary liver cancers that have metastasized are associated with poor outcomes. The underlying mechanisms by which the liver defends against these processes are largely unknown. Prohibitin 1 (PHB1) and methionine adenosyltransferase 1A (MAT1A) are highly expressed in the liver. They positively regulate each other and their deletion results in primary liver cancer. Here we investigated their roles in primary and secondary liver cancer metastasis. METHODS: We identified common target genes of PHB1 and MAT1A using a metastasis array, and measured promoter activity and transcription factor binding using luciferase reporter assays and chromatin immunoprecipitation, respectively. We examined how PHB1 or MAT1A loss promotes liver cancer metastasis and whether their loss sensitizes to colorectal liver metastasis (CRLM). RESULTS: Matrix metalloproteinase-7 (MMP-7) is a common target of MAT1A and PHB1 and its induction is responsible for increased migration and invasion when MAT1A or PHB1 is silenced. Mechanistically, PHB1 and MAT1A negatively regulate MMP7 promoter activity via an AP-1 site by repressing the MAFG-FOSB complex. Loss of MAT1A or PHB1 also increased MMP-7 in extracellular vesicles, which were internalized by colon and pancreatic cancer cells to enhance their oncogenicity. Low hepatic MAT1A or PHB1 expression sensitized to CRLM, but not if endogenous hepatic MMP-7 was knocked down first, which lowered CD4+ T cells while increasing CD8+ T cells in the tumor microenvironment. Hepatocytes co-cultured with colorectal cancer cells express less MAT1A/PHB1 but more MMP-7. Consistently, CRLM raised distant hepatocytes' MMP-7 expression in mice and humans. CONCLUSION: We have identified a PHB1/MAT1A-MAFG/FOSB-MMP-7 axis that controls primary liver cancer metastasis and sensitization to CRLM. IMPACT AND IMPLICATIONS: Primary and secondary liver cancer metastasis is associated with poor outcomes but whether the liver has underlying defense mechanism(s) against metastasis is unknown. Here we examined the hypothesis that hepatic prohibitin 1 (PHB1) and methionine adenosyltransferase 1A (MAT1A) cooperate to defend the liver against metastasis. Our studies found PHB1 and MAT1A form a complex that suppresses matrix metalloproteinase-7 (MMP-7) at the transcriptional level and loss of either PHB1 or MAT1A sensitizes the liver to metastasis via MMP-7 induction. Strategies that target the PHB1/MAT1A-MMP-7 axis may be a promising approach for the treatment of primary and secondary liver cancer metastasis.

Proteomic Analysis of the Supernatant from Bone Marrow Mesenchymal Stem Cells under High Glucose Conditions.

Diabetes mellitus hinders the process of bone regeneration by inhibiting the function of mesenchymal stem cells (MSCs) through elevated glucose levels, thereby impeding osteointegration. The stem cell niche (SCN) plays a crucial role in determining the fate of stem cells by integrating various signals. However, the precise mechanism by which high glucose levels affect the SCN and subsequently influence the function of MSCs remains unclear. In this study, we employed proteomic analysis to identify proteins with altered expression in the extracellular matrix (ECM), aiming to elucidate the underlying mechanism. Three cell supernatants were collected from bone marrow mesenchymal stem cells (BMSCs) or BMSCs stimulated with high glucose (BMSCs+Hg). A total of 590 differentially expressed proteins were identified, which were found to be associated with the ECM, including aging, autophagy, and osteogenic differentiation. The findings of our study indicate that elevated glucose levels exert an influence on the molecular aspects of the SCN, potentially contributing to a better comprehension of the underlying mechanism.

Bomidin attenuates inflammation of periodontal ligament stem cells and periodontitis in mice via inhibiting ferroptosis.

AIM: Periodontitis is a prevalent oral immunoinflammatory condition that is distinguished by the compromised functionality of periodontal ligament stem cells (PDLSCs). Bomidin, a new recombinant antimicrobial peptide (AMP), exhibits antibacterial properties and modulates immune responses. Nevertheless, the precise anti-inflammatory impact of bomidin in periodontitis has yet to be fully elucidated. Thus, the study aimed to clarified the role of bomidin in modulating inflammation and its underlying mechanisms. METHODS: TNF-α was applied to treating PDLSCs for establishing a cell model of periodontitis. Bomidin, RSL3, ML385 and cycloheximide were also used to treat PDLSCs. Transcriptome sequencing, RT-qPCR, western blot, immunofluorescence, immunohistochemistry, Fe2+ detection probe, molecular docking, Co-IP assay, ubiquitination assay and murine models of periodontitis were used. RESULTS: Our study demonstrated that bomidin effectively suppressed inflammation in PDLSCs stimulated by TNF-α, through down-regulating the MAPK and NF-κB signaling pathways. Furthermore, bomidin exerted inhibitory effects on ferroptosis and activated the Keap1/Nrf2 pathway in the TNF-α group. There is a strong likelihood of bonding bomidin with Keap1 protein, which facilitated the degradation of Keap1 protein via the ubiquitin-proteasome pathway, leading to an enhanced translocation of Nrf2 protein to the nucleus. CONCLUSIONS: Bomidin can directly bond to Keap1 protein, resulting in the degradation of Keap1 through the ubiquitin-proteasome pathway, thereby further activating the Keap1/Nrf2 pathway. The upregulation of the Keap1/Nrf2 signaling pathway was found to contribute to the suppression of ferroptosis, ultimately alleviating inflammation in treatment of periodontitis.

CSNK2A1-mediated MAX phosphorylation upregulates HMGB1 and IL-6 expression in cholangiocarcinoma progression.

BACKGROUND: We established a novel diethylnitrosamine (DEN) -induced mouse model that reflected the progression of cholangiocarcinoma (CCA) from atypical cystic hyperplasia. METHODS: BALB/c mice were administered DEN by oral gavage. Cells isolated from livers were analyzed for expression of CSNK2A1, MAX and MAX-interacting proteins. Human CCA cell lines (MzChA-1, HuCCT1), normal human cholangiocyte (H69), human hepatic stellate cells (LX-2), macrophages (RAW 264.7), and primary hepatic cells were used for cellular and molecular biology assays. RESULTS: Expression of MAX, CSNK2A1, C-MYC, ß-catenin, HMGB1, and IL-6 was upregulated in hepatic cells from CCA liver tissue. The half-life of MAX is higher in CCA cells, and this favors their proliferation. Overexpression of MAX increased growth, migration, and invasion of MzChA-1, whereas silencing of MAX had the opposite effect. MAX positively regulated IL-6 and HMGB1 through paracrine signaling in HepG2, LX2, and RAW cells and autocrine signaling in MzChA-1 cells. CSNK2A1-mediated MAX phosphorylation shifts MAX-MAX homodimer to C-MYC-MAX and ß-catenin-MAX heterodimers and increases the HMGB1 and IL-6 promoter activities. Increase of MAX phosphorylation promotes cell proliferation, migration, invasion, and cholangiocarcinogenesis. The casein kinase 2 inhibitor CX-4945 induces cell cycle arrest and inhibits cell proliferation, migration, invasion, and carcinogenesis in MzChA-1 cells through the downregulation of CSNK2A1, MAX, and MAX-interaction proteins. CONCLUSION: C-MYC-MAX and ß-catenin-MAX binding to E-box site or ß-catenin-MAX bound to TCFs/LEF1 enhanced HMGB1 or IL-6 promoter activities, respectively. IL-6 and HMGB1 secreted by hepatocytes, HSCs, and KCs exert paracrine effects on cholangiocytes to promote cell growth, migration, and invasion and lead to the progression of cholangiocarcinogenesis. CX-4945 provides perspectives on therapeutic strategies to attenuate progression from atypical cystic hyperplasia to cholangiocarcinogenesis.

Targeting A-kinase anchoring protein 12 phosphorylation in hepatic stellate cells regulates liver injury and fibrosis in mouse models.

Trans-differentiation of hepatic stellate cells (HSCs) to activated state potentiates liver fibrosis through release of extracellular matrix (ECM) components, distorting the liver architecture. Since limited antifibrotics are available, pharmacological intervention targeting activated HSCs may be considered for therapy. A-kinase anchoring protein 12 (AKAP12) is a scaffolding protein that directs protein kinases A/C (PKA/PKC) and cyclins to specific locations spatiotemporally controlling their biological effects. It has been shown that AKAP12's scaffolding functions are altered by phosphorylation. In previously published work, observed an association between AKAP12 phosphorylation and HSC activation. In this work, we demonstrate that AKAP12's scaffolding activity toward the endoplasmic reticulum (ER)-resident collagen chaperone, heat-shock protein 47 (HSP47) is strongly inhibited by AKAP12's site-specific phosphorylation in activated HSCs. CRISPR-directed gene editing of AKAP12's phospho-sites restores its scaffolding toward HSP47, inhibiting HSP47's collagen maturation functions, and HSC activation. AKAP12 phospho-editing dramatically inhibits fibrosis, ER stress response, HSC inflammatory signaling, and liver injury in mice. Our overall findings suggest a pro-fibrogenic role of AKAP12 phosphorylation that may be targeted for therapeutic intervention in liver fibrosis.

Depletion of mitochondrial methionine adenosyltransferase α1 triggers mitochondrial dysfunction in alcohol-associated liver disease.

MATα1 catalyzes the synthesis of S-adenosylmethionine, the principal biological methyl donor. Lower MATα1 activity and mitochondrial dysfunction occur in alcohol-associated liver disease. Besides cytosol and nucleus, MATα1 also targets the mitochondria of hepatocytes to regulate their function. Here, we show that mitochondrial MATα1 is selectively depleted in alcohol-associated liver disease through a mechanism that involves the isomerase PIN1 and the kinase CK2. Alcohol activates CK2, which phosphorylates MATα1 at Ser114 facilitating interaction with PIN1, thereby inhibiting its mitochondrial localization. Blocking PIN1-MATα1 interaction increased mitochondrial MATα1 levels and protected against alcohol-induced mitochondrial dysfunction and fat accumulation. Normally, MATα1 interacts with mitochondrial proteins involved in TCA cycle, oxidative phosphorylation, and fatty acid ß-oxidation. Preserving mitochondrial MATα1 content correlates with higher methylation and expression of mitochondrial proteins. Our study demonstrates a role of CK2 and PIN1 in reducing mitochondrial MATα1 content leading to mitochondrial dysfunction in alcohol-associated liver disease.

Deregulated 14-3-3ζ and methionine adenosyltransferase α1 interplay promotes liver cancer tumorigenesis in mice and humans.

Methionine adenosyltransferase 1A (MAT1A) is a tumor suppressor downregulated in hepatocellular carcinoma and cholangiocarcinoma, two of the fastest rising cancers worldwide. We compared MATα1 (protein encoded by MAT1A) interactome in normal versus cancerous livers by mass spectrometry to reveal interactions with 14-3-3ζ. The MATα1/14-3-3ζ complex was critical for the expression of 14-3-3ζ. Similarly, the knockdown and small molecule inhibitor for 14-3-3ζ (BV02), and ChIP analysis demonstrated the role of 14-3-3ζ in suppressing MAT1A expression. Interaction between MATα1 and 14-3-3ζ occurs directly and is enhanced by AKT2 phosphorylation of MATα1. Blocking their interaction enabled nuclear MATα1 translocation and inhibited tumorigenesis. In contrast, overexpressing 14-3-3ζ lowered nuclear MATα1 levels and promoted tumor progression. However, tumor-promoting effects of 14-3-3ζ were eliminated when liver cancer cells expressed mutant MATα1 unable to interact with 14-3-3ζ. Taken together, the reciprocal negative regulation that MATα1 and 14-3-3ζ exert is a key mechanism in liver tumorigenesis.

Reciprocal Regulation Between Forkhead Box M1/NF-κB and Methionine Adenosyltransferase 1A Drives Liver Cancer.

BACKGROUND AND AIMS: Forkhead box M1 (FOXM1) and nuclear factor kappa B (NF-ĸB) are oncogenic drivers in liver cancer that positively regulate each other. We showed that methionine adenosyltransferase 1A (MAT1A) is a tumor suppressor in the liver and inhibits NF-ĸB activity. Here, we examined the interplay between FOXM1/NF-κB and MAT1A in liver cancer. APPROACH AND RESULTS: We examined gene and protein expression, effects on promoter activities and binding of proteins to promoter regions, as well as effects of FOXM1 inhibitors T0901317 (T0) and forkhead domain inhibitory-6 (FDI-6) in vitro and in xenograft and syngeneic models of liver cancer. We found, in both hepatocellular carcinoma and cholangiocarcinoma, that an induction in FOXM1 and NF-κB expression is accompanied by a fall in MATα1 (protein encoded by MAT1A). The Cancer Genome Atlas data set confirmed the inverse correlation between FOXM1 and MAT1A. Interestingly, FOXM1 directly interacts with MATα1 and they negatively regulate each other. In contrast, FOXM1 positively regulates p50 and p65 expression through MATα1, given that the effect is lost in its absence. FOXM1, MATα1, and NF-κB all bind to the FOX binding sites in the FOXM1 and MAT1A promoters. However, binding of FOXM1 and NF-κB repressed MAT1A promoter activity, but activated the FOXM1 promoter. In contrast, binding of MATα1 repressed the FOXM1 promoter. MATα1 also binds and represses the NF-κB element in the presence of p65 or p50. Inhibiting FOXM1 with either T0 or FDI-6 inhibited liver cancer cell growth in vitro and in vivo. However, inhibiting FOXM1 had minimal effects in liver cancer cells that do not express MAT1A. CONCLUSIONS: We have found a crosstalk between FOXM1/NF-κB and MAT1A. Up-regulation in FOXM1 lowers MAT1A, but raises NF-κB, expression, and this is a feed-forward loop that enhances tumorigenesis.

The mitochondrial chaperone Prohibitin 1 negatively regulates interleukin-8 in human liver cancers.

Prohibitin 1 (PHB1) is a mitochondrial chaperone whose expression is dysregulated in cancer. In liver cancer, PHB1 acts as a tumor suppressor, but the mechanisms of tumor suppression are incompletely understood. Here we aimed to determine PHB1 target genes to better understand how PHB1 influences liver tumorigenesis. Using RNA-Seq analysis, we found interleukin-8 (IL-8) to be one of the most highly up-regulated genes following PHB1 silencing in HepG2 cells. Induction of IL-8 expression also occurred in multiple liver and nonliver cancer cell lines. We examined samples from 178 patients with hepatocellular carcinoma (HCC) and found that IL-8 mRNA levels were increased, whereas PHB1 mRNA levels were decreased, in the tumors compared with adjacent nontumorous tissues. Notably, HCC patients with high IL-8 expression have significantly reduced survival. An inverse correlation between PHB1 and IL-8 mRNA levels is found in HCCs with reduced PHB1 expression. To understand the molecular basis for these observations, we altered PHB1 levels in liver cancer cells. Overexpression of PHB1 resulted in lowered IL-8 expression and secretion. Silencing PHB1 increased c-Jun N-terminal kinase (JNK) and NF-κB activity, induced nuclear accumulation of c-JUN and p65, and enhanced their binding to the IL-8 promoter containing AP-1 and NF-κB elements. Conditioned medium from PHB1-silenced HepG2 cells increased migration and invasion of parental HepG2 and SK-hep-1 cells, and this was blocked by co-treatment with neutralizing IL-8 antibody. In summary, our findings show that reduced PHB1 expression induces IL-8 transcription by activating NF-κB and AP-1, resulting in enhanced IL-8 expression and release to promote tumorigenesis.

Prohibitin 1 Acts As a Negative Regulator of Wingless/Integrated-Beta-Catenin Signaling in Murine Liver and Human Liver Cancer Cells.

Prohibitin1 (PHB1) is a mitochondrial chaperone with diverse functions that include cell proliferation, apoptosis, and mitochondrial homoeostasis. Liver-specific Phb1 knockout (KO) mice develop spontaneous injury and hepatocellular carcinoma (HCC). Our previous work demonstrated that PHB1 negatively regulates the H19-insulin-like growth factor 2 (IGF2)-H19-IGF2 axis signaling pathway and E-box activity in hepatocytes and HCC cells. Phb1 KO livers exhibited increased expression of multiple wingless/integrated (WNT) target genes compared to control littermates. Therefore, we hypothesized that PHB1 is a negative regulator of WNT-beta-catenin signaling in the liver. Analysis of livers from Phb1 KO mice demonstrated an activation of the WNT-beta-catenin pathway as determined by phosphorylation of glycogen synthase kinase 3 (GSK3)betaserine [Ser]9 and protein kinase B (AKT)Ser473. Phb1 KO livers showed increased messenger RNA (mRNA) levels of multiple WNT ligands, with Wnt7a (79-fold), Wnt10a (12-fold), and Wnt16 (48-fold) being most highly overexpressed compared to control littermates. Subcellular fractionation of liver cells from Phb1 KO mice indicated that hepatocytes are the main source of WNT ligands. Immunostaining and cellular colocalization analysis of Phb1 KO livers demonstrated expression of WNT7a, WNT10a, and WNT16 in hepatocytes. Chromatin immunoprecipitation revealed increased binding of transcription factor E2F1 (E2F1) to the Wnt10a promoter in Phb1 KO livers and WNT9A in HepG2 cells. PHB1 silencing in HepG2 cells activated WNT signaling, whereas its overexpression caused inactivation of this pathway. PHB1 silencing in HepG2 cells induced the expression of multiple WNT ligands of which WNT9A induction was partly regulated through E2F1. Conclusion: PHB1 acts as a negative regulator of WNT signaling, and its down-regulation causes the induction of multiple WNT ligands and downstream activation of canonical WNT-beta-catenin signaling in murine liver and human HCC cells, in part through E2F1.

Mechanisms of MAFG Dysregulation in Cholestatic Liver Injury and Development of Liver Cancer.

BACKGROUND & AIMS: MAF bZIP transcription factor G (MAFG) is activated by the farnesoid X receptor to repress bile acid synthesis. However, expression of MAFG increases during cholestatic liver injury in mice and in cholangiocarcinomas. MAFG interacts directly with methionine adenosyltransferase α1 (MATα1) and other transcription factors at the E-box element to repress transcription. We studied mechanisms of MAFG up-regulation in cholestatic tissues and the pathways by which S-adenosylmethionine (SAMe) and ursodeoxycholic acid (UDCA) prevent the increase in MAFG expression. We also investigated whether obeticholic acid (OCA), an farnesoid X receptor agonist, affects MAFG expression and how it contributes to tumor growth in mice. METHODS: We obtained 7 human cholangiocarcinoma specimens and adjacent non-tumor tissues from patients that underwent surgical resection in California and 113 hepatocellular carcinoma (HCC) specimens and adjacent non-tumor tissues from China, along with clinical data from patients. Tissues were analyzed by immunohistochemistry. MAT1A, MAT2A, c-MYC, and MAFG were overexpressed or knocked down with small interfering RNAs in MzChA-1, KMCH, Hep3B, and HepG2 cells; some cells were incubated with lithocholic acid (LCA, which causes the same changes in gene expression observed during chronic cholestatic liver injury in mice), SAMe, UDCA (100 µM), or farnesoid X receptor agonists. MAFG expression and promoter activity were measured using real-time polymerase chain reaction, immunoblot, and transient transfection. We performed electrophoretic mobility shift, and chromatin immunoprecipitation assays to study proteins that occupy promoter regions. We studied mice with bile-duct ligation, orthotopic cholangiocarcinomas, cholestasis-induced cholangiocarcinoma, diethylnitrosamine-induced liver tumors, and xenograft tumors. RESULTS: LCA activated expression of MAFG in HepG2 and MzChA-1 cells, which required the activator protein-1, nuclear factor-κB, and E-box sites in the MAFG promoter. LCA reduced expression of MAT1A but increased expression of MAT2A in cells. Overexpression of MAT2A increased activity of the MAFG promoter, whereas knockdown of MAT2A reduced it. MAT1A and MAT2A had opposite effects on the activator protein-1, nuclear factor-κB, and E-box-mediated promoter activity. Expression of MAFG and MAT2A increased, and expression of MAT1A decreased, in diethylnitrosamine-induced liver tumors in mice. SAMe and UDCA had shared and distinct mechanisms of preventing LCA-mediated increased expression of MAFG. OCA increased expression of MAFG, MAT2A, and c-MYC, but reduced expression of MAT1A. Incubation of human liver and biliary cancer cells lines with OCA promoted their proliferation; in nude mice given OCA, xenograft tumors were larger than in mice given vehicle. Levels of MAFG were increased in human HCC and cholangiocarcinoma tissues compared with non-tumor tissues. High levels of MAFG in HCC samples correlated with hepatitis B, vascular invasion, and shorter survival times of patients. CONCLUSIONS: Expression of MAFG increases in cells and tissues with cholestasis, as well as in human cholangiocarcinoma and HCC specimens; high expression levels correlate with tumor progression and reduced survival time. SAMe and UDCA reduce expression of MAFG in response to cholestasis, by shared and distinct mechanisms. OCA induces MAFG expression, cancer cell proliferation, and growth of xenograft tumors in mice.

Prohibitin 1 suppresses liver cancer tumorigenesis in mice and human hepatocellular and cholangiocarcinoma cells.

Prohibitin 1 (PHB1) is best known as a mitochondrial chaperone, and its role in cancer is conflicting. Mice lacking methionine adenosyltransferase α1 (MATα1) have lower PHB1 expression, and we reported that c-MYC interacts directly with both proteins. Furthermore, c-MYC and MATα1 exert opposing effects on liver cancer growth, prompting us to examine the interplay between PHB1, MATα1, and c-MYC and PHB1's role in liver tumorigenesis. We found that PHB1 is highly expressed in normal hepatocytes and bile duct epithelial cells and down-regulated in most human hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA). In HCC and CCA cells, PHB1 expression correlates inversely with growth. PHB1 and MAT1A positively regulate each other's expression, whereas PHB1 negatively regulates the expression of c-MYC, MAFG, and c-MAF. Both PHB1 and MATα1 heterodimerize with MAX, bind to the E-box element, and repress E-box promoter activity. PHB1 promoter contains a repressive E-box element and is occupied mainly by MAX, MNT, and MATα1 in nonmalignant cholangiocytes and noncancerous tissues that switched to c-MYC, c-MAF, and MAFG in cancer cells and human HCC/CCA. All 8-month-old liver-specific Phb1 knockout mice developed HCC, and one developed CCA. Five-month-old Phb1 heterozygotes, but not Phb1 flox mice, developed aberrant bile duct proliferation; and one developed CCA 3.5 months after left and median bile duct ligation. Phb1 heterozygotes had a more profound fall in the expression of glutathione synthetic enzymes and higher hepatic oxidative stress following left and median bile duct ligation. CONCLUSION: We have identified that PHB1, down-regulated in most human HCC and CCA, heterodimerizes with MAX to repress the E-box and positively regulates MAT1A while suppressing c-MYC, MAFG, and c-MAF expression; in mice, reduced PHB1 expression predisposes to the development of cholestasis-induced CCA. (Hepatology 2017;65:1249-1266).

Extracellular vesicles released by hepatocytes from gastric infusion model of alcoholic liver disease contain a MicroRNA barcode that can be detected in blood.

Extracellular vesicles (EVs) released during cell stress, or demise, can contain a barcode of the cell origin, including specific microRNAs (miRNAs). Here, we tested the hypothesis that during early alcoholic steatohepatitis (ASH) development, hepatocytes (HCs) release EVs with an miRNA signature that can be measured in circulation. A time-course experiment showed that after 2 weeks of intragastric infusion, a time point that results in isolated steatosis, there was no increase of blood EVs. After 4 weeks of infusion, mice developed features of early ASH accompanied by a marked increase in the level of EVs in blood (P < 0.05), as well as in culture media of isolated HCs (P < 0.001) and hepatic macrophages (P < 0.001), with HCs being the predominant source of EVs. The transcriptome analysis of HC-EVs from ASH mice detected differentially expressed miRNAs, including nine significantly up-regulated and four significantly down-regulated miRNAs. Target prediction and pathway analyses of the up-regulated miRNAs identified 121 potential target genes involved in inflammatory and cancer pathways, such as nuclear factor kappa B, EGF, Wnt, and B-cell lymphoma 2. Three miRNAs, let7f, miR-29a, and miR-340, were increased in blood EVs from ASH mice (P < 0.05), but not in blood EVs from three other models of chronic liver injury, including bile duct ligation, nonalcoholic steatohepatitis, and obese mice, as well as EVs released from hepatocytes exposed to ethanol. Blood EV level (P < 0.01) and three miRNAs (P < 0.05) were significantly increased in patients with ambulatory mild ALD as compared to nonalcoholics. CONCLUSION: Damaged hepatocytes from ASH mice are a key EV source with a specific miRNA cargo, which are specific for ASH-related liver injury. These findings uncover EVs as a potentially novel diagnostic for ASH. (Hepatology 2017;65:475-490).

Activation of hepatic stellate cell in Pten null liver injury model.

BACKGROUND: Hepatic fibrosis is a prominent pathological feature associated with chronic liver disease including non-alcoholic hepatosteatosis (NASH), and a precursor for liver cancer development. We previously reported that PTEN loss in the liver, which leads to hyperactivated liver insulin signaling results in NASH development. Here we used the same mouse model to study the progression from steatosis to fibrosis. RESULTS: The Pten null livers develop progressive liver fibrosis as indicated by Sirius Red staining and increased expression of collagen I, Timp 1, SMAα, and p75NTR. Consistently, hepatic stellate cells (HSCs) isolated from Pten null livers are readily activated when compared with that from mice with intact PTEN. Deletion of AKT2, the downstream target of PTEN signal, blocked NASH development, and alleviated fibrosis. HSCs from the Pten/Akt2 double null mice are quiescent like those isolated from the control livers. Our analysis shows that the activation of HSCs does not depend on the intrinsic signals regulated by PI3K/AKT, the target of PTEN, but does depend on steatosis and injury to the liver. During the progression of liver fibrosis in the Pten null model, Wnt ligands and signaling receptor are induced, concurrent with the reduction of sFRP5, a Wnt antagonist. We showed that treatment of HSCs with Wnt receptor antagonist blocks the observed morphological changes when HSCs undergo activation in culture. This signal appears to be mediated by ß-catenin, as manipulating ß-catenin signaling alters marker gene expressions of HSC activation. CONCLUSIONS: Wnt/ß-catenin activation serves as an important mediator for fibrosis development resulting from NASH using a mouse model where NASH is mimicked by PTEN loss.

Deregulated methionine adenosyltransferase α1, c-Myc, and Maf proteins together promote cholangiocarcinoma growth in mice and humans(‡).

UNLABELLED: c-Myc induction drives cholestatic liver injury and cholangiocarcinoma (CCA) in mice, and induction of Maf proteins (MafG and c-Maf) contributes to cholestatic liver injury, whereas S-adenosylmethionine (SAMe) administration is protective. Here, we determined whether there is interplay between c-Myc, Maf proteins, and methionine adenosyltransferase α1 (MATα1), which is responsible for SAMe biosynthesis in the liver. We used bile duct ligation (BDL) and lithocholic acid (LCA) treatment in mice as chronic cholestasis models, a murine CCA model, human CCA cell lines KMCH and Huh-28, human liver cancer HepG2, and human CCA specimens to study gene and protein expression, protein-protein interactions, molecular mechanisms, and functional outcomes. We found that c-Myc, MATα1 (encoded by MAT1A), MafG, and c-Maf interact with one another directly. MAT1A expression fell in hepatocytes and bile duct epithelial cells during chronic cholestasis and in murine and human CCA. The opposite occurred with c-Myc, MafG, and c-Maf expression. MATα1 interacts mainly with Mnt in normal liver, but this switches to c-Maf, MafG, and c-Myc in cholestatic livers and CCA. Promoter regions of these genes have E-boxes that are bound by MATα1 and Mnt in normal liver and benign bile duct epithelial cells that switched to c-Myc, c-Maf, and MafG in cholestasis and CCA cells. E-box positively regulates c-Myc, MafG, and c-Maf, but it negatively regulates MAT1A. MATα1 represses, whereas c-Myc, MafG, and c-Maf enhance, E-box-driven promoter activity. Knocking down MAT1A or overexpressing MafG or c-Maf enhanced CCA growth and invasion in vivo. CONCLUSION: There is a novel interplay between MATα1, c-Myc, and Maf proteins, and their deregulation during chronic cholestasis may facilitate CCA oncogenesis. (Hepatology 2016;64:439-455).

Low intracellular iron increases the stability of matriptase-2.

Matriptase-2 (MT2) is a type II transmembrane serine protease that is predominantly expressed in hepatocytes. It suppresses the expression of hepatic hepcidin, an iron regulatory hormone, by cleaving membrane hemojuvelin into an inactive form. Hemojuvelin is a bone morphogenetic protein (BMP) co-receptor. Here, we report that MT2 is up-regulated under iron deprivation. In HepG2 cells stably expressing the coding sequence of the MT2 gene, TMPRSS6, incubation with apo-transferrin or the membrane-impermeable iron chelator, deferoxamine mesylate salt, was able to increase MT2 levels. This increase did not result from the inhibition of MT2 shedding from the cells. Rather, studies using a membrane-permeable iron chelator, salicylaldehyde isonicotinoyl hydrazone, revealed that depletion of cellular iron was able to decrease the degradation of MT2 independently of internalization. We found that lack of the putative endocytosis motif in its cytoplasmic domain largely abolished the sensitivity of MT2 to iron depletion. Neither acute nor chronic iron deficiency was able to alter the association of Tmprss6 mRNA with polyribosomes in the liver of rats indicating a lack of translational regulation by low iron levels. Studies in mice showed that Tmprss6 mRNA was not regulated by iron nor the BMP-mediated signaling with no evident correlation with either Bmp6 mRNA or Id1 mRNA, a target of BMP signaling. These results suggest that regulation of MT2 occurs at the level of protein degradation rather than by changes in the rate of internalization and translational or transcriptional mechanisms and that the cytoplasmic domain of MT2 is necessary for its regulation.

Mesodermal mesenchymal cells give rise to myofibroblasts, but not epithelial cells, in mouse liver injury.

UNLABELLED: Hepatic stellate cells (HSCs) and portal fibroblasts (PFs) are believed to be the major source of myofibroblasts that participate in fibrogenesis by way of synthesis of proinflammatory cytokines and extracellular matrices. Previous lineage tracing studies using MesP1(Cre) and Rosa26lacZ(flox) mice demonstrated that MesP1+ mesoderm gives rise to mesothelial cells (MCs), which differentiate into HSCs and PFs during liver development. In contrast, several in vivo and in vitro studies reported that HSCs can differentiate into other cell types, including hepatocytes, cholangiocytes, and progenitor cell types known as oval cells, thereby acting as stem cells in the liver. To test whether HSCs give rise to epithelial cells in adult liver, we determined the hepatic lineages of HSCs and PFs using MesP1(Cre) and Rosa26mTmG(flox) mice. Genetic cell lineage tracing revealed that the MesP1+ mesoderm gives rise to MCs, HSCs, and PFs, but not to hepatocytes or cholangiocytes, in the adult liver. Upon carbon tetrachloride injection or bile duct ligation surgery-mediated liver injury, mesodermal mesenchymal cells, including HSCs and PFs, differentiate into myofibroblasts but not into hepatocytes or cholangiocytes. Furthermore, differentiation of the mesodermal mesenchymal cells into oval cells was not observed. These results indicate that HSCs are not sufficiently multipotent to produce hepatocytes, cholangiocytes, or oval cells by way of mesenchymal-epithelial transition in vivo. CONCLUSION: Cell lineage tracing demonstrated that mesodermal mesenchymal cells including HSCs are the major source of myofibroblasts but do not differentiate into epithelial cell types such as hepatocytes, cholangiocytes, and oval cells.

Increased iron loading induces Bmp6 expression in the non-parenchymal cells of the liver independent of the BMP-signaling pathway.

Bone morphogenetic protein 6 (BMP6) is an essential cytokine for the expression of hepcidin, an iron regulatory hormone secreted predominantly by hepatocytes. Bmp6 expression is upregulated by increased iron-levels in the liver. Both hepatocytes and non-parenchymal liver cells have detectable Bmp6 mRNA. Here we showed that induction of hepcidin expression in hepatocytes by dietary iron is associated with an elevation of Bmp6 mRNA in the non-parenchymal cells of the liver. Consistently, incubation with iron-saturated transferrin induces Bmp6 mRNA expression in isolated hepatic stellate cells, but not in hepatocytes. These observations suggest an important role of the non-parenchymal liver cells in regulating iron-homeostasis by acting as a source of Bmp6.

Mesothelial cells give rise to hepatic stellate cells and myofibroblasts via mesothelial-mesenchymal transition in liver injury.

In many organs, myofibroblasts play a major role in the scarring process in response to injury. In liver fibrogenesis, hepatic stellate cells (HSCs) are thought to transdifferentiate into myofibroblasts, but the origins of both HSCs and myofibroblasts remain elusive. In the developing liver, lung, and intestine, mesothelial cells (MCs) differentiate into specific mesenchymal cell types; however, the contribution of this differentiation to organ injury is unknown. In the present study, using mouse models, conditional cell lineage analysis has demonstrated that MCs expressing Wilms tumor 1 give rise to HSCs and myofibroblasts during liver fibrogenesis. Primary MCs, isolated from adult mouse liver using antibodies against glycoprotein M6a, undergo myofibroblastic transdifferentiation. Antagonism of TGF-ß signaling suppresses transition of MCs to mesenchymal cells both in vitro and in vivo. These results indicate that MCs undergo mesothelial-mesenchymal transition and participate in liver injury via differentiation to HSCs and myofibroblasts.